Aging and insulin resistance have been related to reduced mitochondrial function and oxidative stress. Muscular phosphodiesters (PDE) are comprised of metabolites of phospholipid breakdown and may reflect membrane damage. We aimed to test the hypothesis that myocellular PDE are increased in patients with type 2 diabetes (T2D) and correlate inversely with mitochondrial ATP turnover.
A Cross-sectional study in the Clinical Research Facility of an University hospital was performed. 10 nonobese middle-aged patients with T2D, 10 healthy humans matched for sex, age and physical activity index (CONm) and 18 young healthy humans (CONy) were included. Myocellular PDE and unidirectional flux through ATP synthase (fATP) were measured with 31P magnetic resonance spectroscopy (MRS). Intramyocellular (IMCL) and hepatocellular lipid deposition (HCL) were quantified with 1H MRS. Insulin sensitivity (Rd) was assessed from hyperinsulinemic-euglycemic clamp tests in 10 T2D, 10 CONm and 11 CONy.
During fasting, T2D and CONm had 1.5 fold greater PDE than CONy (2.8±0.2, 2.5±0.2, 1.7±0.1 mmol/l, P = 0.004). Stimulation by insulin did not affect PDE in any group. PDE correlated negatively with Rd (r = −0.552, p<0.005) and fATP (r = −0.396, p<0.05) and positively with age (r = 0.656, p<0.001) and body mass (r = 0.597, p<0.001). PDE also related positively to HbA1c (r = 0.674, p<0.001) and fasting plasma glucose (r = 0.629, p<0.001) within T2D and across all participants.
Muscular PDE concentrations associate with age, lower resting mitochondrial activity and insulin resistance, which is determined mainly by body mass and glycemia.
Citation: Szendroedi J, Schmid AI, Chmelik M, Krssak M, Nowotny P, Prikoszovich T, et al. (2011) Skeletal Muscle Phosphodiester Content Relates to Body Mass and Glycemic Control. PLoS ONE 6(7): e21846. doi:10.1371/journal.pone.0021846
Editor: Ian Lanza, Mayo Clinic, United States of America
Received: April 28, 2011; Accepted: June 7, 2011; Published: July 14, 2011
Copyright: © 2011 Szendroedi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: European Foundation for the Study of Diabetes (EFSD; Lilly-supported grant), the Schmutzler Stiftung, the Skröder Stiftung, the German Diabetes Foundation (DFG; SFB 575) and the German Center for Diabetes Research (DZD e.V.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Type 2 diabetes mellitus (T2D) represents one of the world's greatest economic and health care challenges. T2D results from an imbalance between insulin responsiveness and insulin secretion. Skeletal muscle is mainly responsible for whole-body insulin resistance and determines substrate oxidation particularly during exercise. Reduced mitochondrial oxidative capacity, activity and/or content have been related to aging, intramyocelluar lipid content (IMCL) and muscular insulin resistance , , , .
It has been hypothesized that aging-associated insulin resistance results from cumulative free radical damage leading to lower mitochondrial function and increased IMCL . Of note, preventing oxidative damage by overexpression of antioxidant defense mechanisms indeed protected rodent models from lipid-induced and age-associated insulin resistance , . However, a causal relationship between aging, mitochondria and the development of T2D has been questioned , , . Of note, there is no evidence for disruption of the cellular integrity and contractile function in skeletal muscle of T2D, while patients with myopathies can exhibit mitochondrial abnormalities and insulin resistance , , .
Membrane phospholipids are hydrolyzed to free fatty acids (FFA) and phosphodiesters (PDE) which are regarded as cell membrane degradation products . Myocellular PDE are elevated in patients with myopathies ,  and in elderly ,  and increase in response to treatment with statins . PDE may reflect fiber atrophy, accumulated sarcolemmal damage  or structural and functional changes during loss of muscle mass possibly resulting from cumulative oxidative damage . Thus, PDE may serve as marker of biomembrane integrity , . The relationship between PDE content and mitochondrial function and insulin sensitivity in healthy middle-aged humans and in patients with T2D has not yet been reported.
We hypothesized that T2D have elevated myocellular PDE which relates to age, mitochondrial activity and insulin sensitivity. Thus, we measured PDE, unidirectional flux through ATP synthase (fATP) during fasting and insulin stimulation as a marker of resting mitochondrial activity , , insulin stimulated glucose-6-phosphate (ΔG6P) as a marker of insulin-stimulated glucose transport/phosphorylation, whole-body glucose disposal (Rd) and endogenous glucose production (EGP) in T2D, non-diabetic age-matched (CONm) and younger humans (CONy).
All studies were carried out in accordance with the most-recent version of the Declaration of Helsinki and approved by the local ethics committee of the Medical University of Vienna. Informed written consent has been obtained from all participants.
Ten T2D, 10 CONm matched for sex, age and physical activity and 18 CONy were included. PDE content in all participants from one study  and 7 controls from another study  are reported. They underwent complete medical history, clinical examination and lab tests. All participants had comparable physical activity according to Baeckés questionnaire, refrained from any physical exercise for three days and fasted for 12 h before the study. T2D had neither islet cell antibodies nor signs of neurological disorders including diabetes-related neuropathy. Only sulfonylurea and/or metformin were allowed as glucose-lowering medication but withdrawn three days before the study. Four T2D and none of the healthy participants received statins, which were not withdrawn before the study. Control subjects had no family history of T2D.
After baseline blood sampling, D-[6,6-2H2]glucose (98% enriched; Cambridge Isotope Laboratories, http://www.isotope.com/cil/index.cfm) was given as primed-continuous infusion [0 min to 5 min: 3.6 mg.(kg body weight)−1.(fasting plasma glucose in mg/dl)/(90 mg/dl); −115 min to +240 min 0.036 mg.min−1.(kg body weight)−1] to assess Rd and EGP from +220 to +240 min. From 0 to +240 min, hyperinsulinemic-euglycemic clamp tests were performed in 10 T2DM, 10 CONm and 11 CONy, while 7 CONy were only examined at baseline. Insulin (Actrapid; Novo, Bagsvaerd, Denmark) was administered as primed-continuous infusion [40 mU.(m body surface area)−2.min−1] and plasma glucose was controlled by a variable 20% dextrose infusion, 2% enriched with D-[6,6-2H2]glucose according to the hot-glucose-infusion protocol.
IMCL, PDE and other phosphorus metabolites were measured at baseline and during insulin stimulation.
Magnetic Resonance Spectroscopy (MRS)
Measurements were performed on subjects lying supine inside a 3-Tesla MR spectrometer (Medspec S300-DBX; Bruker, Ettlingen, Germany). The right lower leg positioned on a 10-cm circular double resonant 1H/31P surface coil (125.6/50.8 MHz) so that the isocenter of the magnetic field was placed ∼2 cm into the medial head of the gastrocnemius muscle . Phosphorus compounds were measured from the ratio of the integrated peak intensities and ß-ATP resonance intensity in spectra without inversion and saturation (pulse length 150 µs/90°, 2k data, 8 averages, repetition time of 15 s) assuming constant ATP concentrations of 5.5 mmol/l muscle . In human skeletal muscle, the peak between inorganic phosphate (Pi) and phosphocreatine (PCr) represents the signal arising from PDE which is mostly attributed to glycerophospho-ethanolamine/-choline, membrane-bound phospholipid metabolites (Figure 1) . Measurement of PDE has been validated against chemical analysis employing preparations of muscular ethanol-soluble PDE and exogenous glycerol-3-phosphorylcholine, which confirmed that MRS-measured PDE represent the peak resonating at 0.13 ppm .
Phosphorus compounds were measured from the ratio of the integrated respective peak intensities and ß-ATP resonance intensity in spectra without inversion and saturation (pulse length 150 µs/90°, 2k data, 8 averages, repetition time of 15 s) assuming a constant ATP concentration of 5.5 mmol/l muscle.
Myocellular fATP [µmol.(g muscle −1).min−1] was measured with 31P MRS employing the saturation transfer experiment to the Pi/ATP exchange , . Glucose transport/-phosphorylation was assessed from the increase in G6P during the clamp (ΔG6P). IMCL in soleus muscle and liver fat content (HCL) were measured by 1H MRS , .
Plasma glucose was assessed by the glucose oxidase method (Beckman, Fullerton, CA), FFA microfluorimetrically (Wako Chem. USA Inc., Richmond, VA) and plasma insulin by double-antibody radioimmunoassay (Pharmacia, Uppsala, Sweden). All other measurements were assessed in the routine lab.
Calculations and statistics
At baseline, rates of glucose appearance (Ra) were calculated by dividing the tracer D-[6,6- 2H2]glucose infusion rate times tracer enrichment by the percent of tracer enrichment in plasma and subtracting the tracer infusion rate . During the clamp, Ra was calculated using Steele's non-steady state equations . EGP is given as the difference between Ra and mean glucose infusion rates.
Group data are presented as means and SD (Text) or SEM (Figures) and compared with ANOVA and Tukey post-hoc testing as appropriate. Within-group differences were assessed with two-tailed t-tests. Linear correlations are Pearson product-moment correlations. Partial correlation analysis was performed to study the linear relationship between PDE and other variables after excluding the effect of age, BMI or HbA1c. Differences were considered significant at the 5-% level.
All groups were matched for sex (50% female). BMI tended to be (T2D: 27±3; CONm: 26±3; CONy: 24±2 kg/m2) and waist circumference was higher in T2D and CONm (97±9; 92±17; 80±8 cm, p<0.05 T2D vs. CONy). T2D and CONm were also matched for age (59±6; 57±7; 29±5 years, p<0.001 both vs. CONy). T2D had greater fasting plasma glucose (8.9±1.7; 5.2±0.4; 4.8±0.7 mmol/l, p<0.001 vs. both CON groups) and HbA1c (6.9±0.7; 5.5±0.3; 5.2±0.2 mmol/l, p<0.001 vs. both CON), but comparable plasma FFA (0.57±0.11; 0.44±0.22; 0.48±0.26 mmol/l) and low-density lipoprotein (LDL: 3.4±0.8; 3.8±0.9; 2.6±0.5 mmol/l).
During clamp steady-state (220–240 min), mean plasma glucose, insulin and FFA were 5.5±0.5 mmol/l, 514±96 pmol/l and 0.02±0.01 mmol/l without differences between groups. T2D had ∼33% and ∼54% lower Rd than CONm and CONy; CONm had ∼31% lower Rd than CONy (5.7±0.5, 8.5±0.8, 12.4±1.0 mg.kg−1.min−1, p<0.05, p<0.001 T2D vs. CONm and CONy, p<0.005 CONm vs CONy). Insulin-suppressed EGP reflecting hepatic insulin resistance was greater in T2D and CONm (0.23±0.05; 0.22±0.09; −0.14±0.09 mg.kg−1.min−1, p<0.05 both vs. CONy).
Resting mitochondrial activity (fATP) was lower in T2D than in CONy but comparable to CONm . Stimulation by insulin increased fATP in CONm and CONy but not in T2D and unmasked impaired mitochondrial adaptation compared to CONm .
In the fasted state, myocellular PDE contents were ∼65% and ∼47% higher in T2D and CONm compared to CONy, but not different between T2D and CONm (Figure 2A). IMCL and ΔG6P were comparable in all groups as reported  and did not relate to PDE. Insulin stimulation did not affect PDE (basal vs. insulin-stimulated: 2.8±0.8 vs. 2.9±0.7; 2.5±0.7 vs. 2.5±0.7; CONy: 1.8±0.3 vs. 1.8±0.3 mmol/l), which were ∼55% and ∼38% higher in T2D and CONm compared to CONy (p<0.001, p<0.05, Figure 2B). Changes in PDE did not relate to ΔG6P.
PDE correlated negatively with fasting fATP (Figure 3A) and insulin-mediated Rd (Figure 3B), but not with suppressed EGP or insulin-stimulated fATP. PDE related positively to BMI (Figure 3C), waist circumference (r = 0.668, p<0.001), age (r = 0.656, p<0.001), HCL (r = 0.446, p<0.01), HbA1c (Figure 3D) and to fasting plasma glucose (r = 0.629, p<0.001) across all participants and within T2D (r = 0.652, r = 0.653, p<0.05). There was no correlation between muscle PDE and HbA1c in the pooled group of non-diabetic subjects. PDE neither related to physical activity nor to plasma FFA, LDL or IMCL. Correlations of PDE with fATP and Rd were abolished after selective adjustment for either age, BMI or HbA1c.
Myocellular PDE levels are higher in middle-aged patients with T2D and in age-matched controls than in young non-diabetic humans. The relationships of PDE with insulin resistance and mitochondrial function seem to depend mainly on aging, body mass, but also glycemia.
The greater PDE in the middle-aged humans with or without T2D is in accordance with its positive relationship to age as reported previously , but confounding factors such as insulin resistance or mitochondrial function were not identified before . Aging-related alterations of skeletal muscle could simply result from reduced voluntary physical activity, but PDE - in line with previous data  - did not relate to physical activity index. All groups of the present study were sedentary and matched for physical activity, but exercise testing was not performed. PDE has been shown to be increased in professional road cyclists , but to be significantly lower in sprinters than in sedentary and long-distance runners . Accordingly, a large PDE signal has been reported as a sign of a larger relative content of type 1 fibers, i. e. fast-to-slow twitch muscle transformation . However, this observation does not fit with increased PDE in aging or diabetic subjects, who rather have increased , ,  or normal fast glycolytic fiber fractions , . Also, statins have been shown to increase muscle PDE contents , but only four of our patients with T2D were on statins and their PDE content was not different from the other patients. Alternatively, aging-associated cumulative oxidative damage may affect mitochondrial DNA and function and lead to muscular degenerative processes . Our T2D showed lower fATP than both control groups during insulin stimulation, while resting fATP was similarly lower in T2D and CONm than in CONy .
In vivo and ex vivo measures reported lower oxidative capacity in exercising and resting muscle of T2D patients which result from intrinsic impairment and lower mitochondrial content , , , , . fATP, reflecting demand-driven resting mitochondrial activity, is lower in insulin-resistant elderly ,  and non-diabetic offspring of T2D patients , but can be normal in T2D compared to carefully matched non-diabetic humans , . Alternatively, abnormalities in insulin signaling may be the primary events leading to impaired mitochondrial function, or both phenomena may be mutually interrelated . Of note, lower mitochondrial content could be compensated for by greater mitochondrial activity suggesting that insulin sensitivity and mitochondrial function are not uniformly coupled , , , . Nevertheless, partial correlation analysis revealed that PDE contents relate to aging independently of BMI and physical activity, but not independently of fATP and insulin sensitivity. Thus, the impact of aging on PDE is likely mediated by insulin sensitivity and fATP.
Partial correlation analysis further identified PDE contents as an independent indicator of glucometabolic control (HbA1c and fasting plasma glucose) in T2D. Elevated PDE as observed in muscle damage , pain syndromes  and muscle dystrophies, has been previously attributed to oxidative stress , , . One might therefore speculate that glucotoxicity-induced chronic oxidative stress could contribute to the greater PDE as well as to the lower fATP and insulin sensitivity in our T2D. This study found a weak negative relationship between muscular PDE and fATP, which disappeared upon adjusting for age, BMI and HbA1c. While this does not exclude any role of mitochondrial damage for raising PDE levels, it makes such mechanism rather unlikely. However, fATP is only one feature of mitochondrial function and in the present study mitochondrial content and oxidative capacity were not measured. Furthermore, fATP as assessed from the ATP saturation transfer experiment with 31P MRS, reflects in vivo ATP synthase flux resulting from basal energy demand and supplying processes and is therefore a measure of resting mitochondrial activity. .
Muscular PDE related negatively to whole-body glucose disposal but not specifically to insulin-mediated glucose transport/phosphorylation and did not change during insulin stimulation even in insulin sensitive humans. While PDE do not seem to directly reflect insulin-dependent metabolic processes, its tight relationship with BMI points to obesity-associated alterations such as fatty acid composition of membrane phospholipids which may influence insulin binding and action . While lipid-induced insulin resistance generally arises from increased plasma FFA and intracellular lipids , this study found no association between PDE, FFA and IMCL. Nevertheless, PDE are generated by increased activities of phospholipases  which also yield ceramides and diacylglycerols (DAG), possible mediators of insulin resistance , .
Some limitations need to be taken into account. First, no biopsies were taken so that identification of individual components of the PDE peak is not possible. However, the analysis of individual compounds contributing to the PDE signal is difficult, because ischemia during tissue handling will impact on analysis of extracted tissue . Second, no indicator of oxidative stress was determined to analyse the possible role of PDE contents as a biomarker of oxidative stress. However, available markers of oxidative stress rely on various assumptions and do not offer direct measures , . Finally, our T2D cohort comprised of normal weight (n = 4), overweight (n = 4) and obese (n = 2) Caucasians yielding a mean BMI of 27 kg/m2, which was not significantly different but tended to be higher than in controls. While T2D cohorts mostly have mean BMI exceeding 30 kg/m2, other T2D cohorts have lower mean BMI values, e. g. 28.5 kg/m2 in the ADVANCE study . Thus, our T2D group might be suitable and representative to analyse the impact of T2D per se on PDE contents.
In conclusion, muscular PDE are higher in middle-aged patients with or without T2D than in young non-diabetic humans. Body mass and glycemia mainly determine the correlations of PDE with resting mitochondrial activity, insulin resistance and age.
Conceived and designed the experiments: JS AIS WW MR. Performed the experiments: JS AIS MC MK TP AK-W PN. Analyzed the data: JS AIS MC MK TP AK-W PN. Contributed reagents/materials/analysis tools: MW WW MR. Wrote the paper: JS MR. Revised the manuscript: MK WW MW AK-W TP.
- 1. Phielix E, Szendroedi J, Roden M (2010) Mitochondrial Function and Insulin Resistance during Aging - A Mini-Review. Gerontology.
- 2. Lowell BB, Shulman GI (2005) Mitochondrial dysfunction and type 2 diabetes. Science 307: 384–387.
- 3. Schrauwen-Hinderling VB, Kooi ME, Hesselink MK, Jeneson JA, Backes WH, et al. (2007) Impaired in vivo mitochondrial function but similar intramyocellular lipid content in patients with type 2 diabetes mellitus and BMI-matched control subjects. Diabetologia 50: 113–120.
- 4. Phielix E, Schrauwen-Hinderling VB, Mensink M, Lenaers E, Meex R, et al. (2008) Lower intrinsic ADP-stimulated mitochondrial respiration underlies in vivo mitochondrial dysfunction in muscle of male type 2 diabetic patients. Diabetes 57: 2943–2949.
- 5. Lee HY, Choi CS, Birkenfeld AL, Alves TC, Jornayvaz FR, et al. (2010) Targeted expression of catalase to mitochondria prevents age-associated reductions in mitochondrial function and insulin resistance. Cell Metab 12: 668–674.
- 6. Anderson EJ, Lustig ME, Boyle KE, Woodlief TL, Kane DA, et al. (2009) Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans. J Clin Invest.
- 7. Holloszy JO (2009) Skeletal muscle “mitochondrial deficiency” does not mediate insulin resistance. Am J Clin Nutr 89: 463S–466S.
- 8. Karakelides H, Irving BA, Short KR, O'Brien P, Nair KS (2009) Age, obesity, and sex effects on insulin sensitivity and skeletal muscle mitochondrial function. Diabetes.
- 9. Boschmann M, Engeli S, Moro C, Luedtke A, Adams F, et al. (2010) LMNA mutations, skeletal muscle lipid metabolism, and insulin resistance. J Clin Endocrinol Metab 95: 1634–1643.
- 10. Velho G, Byrne MM, Clement K, Sturis J, Pueyo ME, et al. (1996) Clinical phenotypes, insulin secretion, and insulin sensitivity in kindreds with maternally inherited diabetes and deafness due to mitochondrial tRNALeu(UUR) gene mutation. Diabetes 45: 478–487.
- 11. Szendroedi J, Ingo Schmid A, Meyerspeer M, Cervin C, Kacerovsky M, et al. (2009) Impaired mitochondrial function and insulin resistance of skeletal muscle in mitochondrial diabetes. Diabetes Care.
- 12. Cox IJ, Menon DK, Sargentoni J, Bryant DJ, Collins AG, et al. (1992) Phosphorus-31 magnetic resonance spectroscopy of the human liver using chemical shift imaging techniques. J Hepatol 14: 265–275.
- 13. Matthews PM, Allaire C, Shoubridge EA, Karpati G, Carpenter S, et al. (1991) In vivo muscle magnetic resonance spectroscopy in the clinical investigation of mitochondrial disease. Neurology 41: 114–120.
- 14. Edwards RH, Dawson MJ, Wilkie DR, Gordon RE, Shaw D (1982) Clinical use of nuclear magnetic resonance in the investigation of myopathy. Lancet 1: 725–731.
- 15. Younkin DP, Berman P, Sladky J, Chee C, Bank W, et al. (1987) 31P NMR studies in Duchenne muscular dystrophy: age-related metabolic changes. Neurology 37: 165–169.
- 16. Satrustegui J, Berkowitz H, Boden B, Donlon E, McLaughlin A, et al. (1988) An in vivo phosphorus nuclear magnetic resonance study of the variations with age in the phosphodiester content of human muscle. Mech Ageing Dev 42: 105–114.
- 17. Slade JM, Delano MC, Meyer RA (2006) Elevated skeletal muscle phosphodiesters in adults using statin medications. Muscle Nerve 34: 782–784.
- 18. Taylor DJ, Kemp GJ, Thompson CH, Radda GK (1997) Ageing: effects on oxidative function of skeletal muscle in vivo. Mol Cell Biochem 174: 321–324.
- 19. Lanza IR, Nair KS (2010) Mitochondrial function as a determinant of life span. Pflugers Arch 459: 277–289.
- 20. Waters DL, Brooks WM, Qualls CR, Baumgartner RN (2003) Skeletal muscle mitochondrial function and lean body mass in healthy exercising elderly. Mech Ageing Dev 124: 301–309.
- 21. Petersen KF, Befroy D, Dufour S, Dziura J, Ariyan C, et al. (2003) Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science 300: 1140–1142.
- 22. Szendroedi J, Schmid AI, Chmelik M, Toth C, Brehm A, et al. (2007) Muscle mitochondrial ATP synthesis and glucose transport/phosphorylation in type 2 diabetes. PLoS Med 4: e154.
- 23. Prikoszovich T, Winzer C, Schmid AI, Szendroedi J, Chmelik M, et al. (2010) Body and Liver Fat Mass Rather Than Muscle Mitochondrial Function Determines Glucose Metabolism in Women with a History of Gestational Diabetes. Diabetes Care.
- 24. Burt CT, Glonek T, Barany M (1976) Phosphorus-31 nuclear magnetic resonance detection of unexpected phosphodiesters in muscle. Biochemistry 15: 4850–4853.
- 25. Brown TR, Ugurbil K, Shulman RG (1977) 31P nuclear magnetic resonance measurements of ATPase kinetics in aerobic Escherichia coli cells. Proc Natl Acad Sci U S A 74: 5551–5553.
- 26. Krssak M, Falk Petersen K, Dresner A, DiPietro L, Vogel SM, et al. (1999) Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: a 1H NMR spectroscopy study. Diabetologia 42: 113–116.
- 27. Mandarino LJ, Consoli A, Jain A, Kelley DE (1993) Differential regulation of intracellular glucose metabolism by glucose and insulin in human muscle. Am J Physiol 265: E898–905.
- 28. Hother-Nielsen O, Vaag A, Skott P, Beck-Nielsen H (1993) Effect of hyperglycemia per se on glucose turnover rates in patients with insulin-dependent diabetes. Metabolism 42: 86–93.
- 29. Jubrias SA, Bennett RM, Klug GA (1994) Increased incidence of a resonance in the phosphodiester region of 31P nuclear magnetic resonance spectra in the skeletal muscle of fibromyalgia patients. Arthritis Rheum 37: 801–807.
- 30. Hug F, Bendahan D, Le Fur Y, Cozzone PJ, Grelot L (2005) Metabolic recovery in professional road cyclists: a 31P-MRS study. Med Sci Sports Exerc 37: 846–852.
- 31. Bernus G, Gonzalez de Suso JM, Alonso J, Martin PA, Prat JA, et al. (1993) 31P-MRS of quadriceps reveals quantitative differences between sprinters and long-distance runners. Med Sci Sports Exerc 25: 479–484.
- 32. Burt CT, Pluskal MG, Sreter FA (1982) Generation of phosphodiesters during fast-to-slow muscle transformation. A 31P-NMR study. Biochim Biophys Acta 721: 492–494.
- 33. Oberbach A, Bossenz Y, Lehmann S, Niebauer J, Adams V, et al. (2006) Altered fiber distribution and fiber-specific glycolytic and oxidative enzyme activity in skeletal muscle of patients with type 2 diabetes. Diabetes Care 29: 895–900.
- 34. Hickey MS, Carey JO, Azevedo JL, Houmard JA, Pories WJ, et al. (1995) Skeletal muscle fiber composition is related to adiposity and in vitro glucose transport rate in humans. Am J Physiol 268: E453–457.
- 35. Simoneau JA, Veerkamp JH, Turcotte LP, Kelley DE (1999) Markers of capacity to utilize fatty acids in human skeletal muscle: relation to insulin resistance and obesity and effects of weight loss. Faseb J 13: 2051–2060.
- 36. Kelley DE, Simoneau JA (1994) Impaired free fatty acid utilization by skeletal muscle in non-insulin-dependent diabetes mellitus. J Clin Invest 94: 2349–2356.
- 37. He J, Watkins S, Kelley DE (2001) Skeletal muscle lipid content and oxidative enzyme activity in relation to muscle fiber type in type 2 diabetes and obesity. Diabetes 50: 817–823.
- 38. Hebert SL, Lanza IR, Nair KS (2010) Mitochondrial DNA alterations and reduced mitochondrial function in aging. Mech Ageing Dev 131: 451–462.
- 39. Kelley DE, He J, Menshikova EV, Ritov VB (2002) Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes. Diabetes 51: 2944–2950.
- 40. Ritov VB, Menshikova EV, Azuma K, Wood R, Toledo FG, et al. Deficiency of electron transport chain in human skeletal muscle mitochondria in type 2 diabetes mellitus and obesity. Am J Physiol Endocrinol Metab 298: E49–58.
- 41. Boushel R, Gnaiger E, Schjerling P, Skovbro M, Kraunsoe R, et al. (2007) Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle. Diabetologia 50: 790–796.
- 42. 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.
- 43. Hey-Mogensen M, Hojlund K, Vind BF, Wang L, Dela F, et al. (2010) Effect of physical training on mitochondrial respiration and reactive oxygen species release in skeletal muscle in patients with obesity and type 2 diabetes. Diabetologia 53: 1976–1985.
- 44. Sleigh A, Raymond-Barker P, Thackray K, Porter D, Hatunic M, et al. (2011) Mitochondrial dysfunction in patients with primary congenital insulin resistance. J Clin Invest.
- 45. Lefort N, Glancy B, Bowen B, Willis WT, Bailowitz Z, et al. (2010) Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle. Diabetes 59: 2444–2452.
- 46. Pospisilik JA, Knauf C, Joza N, Benit P, Orthofer M, et al. (2007) Targeted deletion of AIF decreases mitochondrial oxidative phosphorylation and protects from obesity and diabetes. Cell 131: 476–491.
- 47. Nair KS, Bigelow ML, Asmann YW, Chow LS, Coenen-Schimke JM, et al. (2008) Asian Indians have enhanced skeletal muscle mitochondrial capacity to produce ATP in association with severe insulin resistance. Diabetes 57: 1166–1175.
- 48. Schunk K, Pitton M, Duber C, Kersjes W, Schadmand-Fischer S, et al. (1999) Dynamic phosphorus-31 magnetic resonance spectroscopy of the quadriceps muscle: effects of age and sex on spectroscopic results. Invest Radiol 34: 116–125.
- 49. Kemp GJ (2008) The interpretation of abnormal 31P magnetic resonance saturation transfer measurements of Pi/ATP exchange in insulin-resistant skeletal muscle. Am J Physiol Endocrinol Metab 294: E640–642. author reply E643–644.
- 50. Pan DA, Lillioja S, Milner MR, Kriketos AD, Baur LA, et al. (1995) Skeletal muscle membrane lipid composition is related to adiposity and insulin action. J Clin Invest 96: 2802–2808.
- 51. Roden M, Price TB, Perseghin G, Petersen KF, Rothman DL, et al. (1996) Mechanism of free fatty acid-induced insulin resistance in humans. J Clin Invest 97: 2859–2865.
- 52. Holland WL, Brozinick JT, Wang LP, Hawkins ED, Sargent KM, et al. (2007) Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance. Cell Metab 5: 167–179.
- 53. Itani SI, Ruderman NB, Schmieder F, Boden G (2002) Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IkappaB-alpha. Diabetes 51: 2005–2011.
- 54. Ristow M, Zarse K, Oberbach A, Kloting N, Birringer M, et al. (2009) Antioxidants prevent health-promoting effects of physical exercise in humans. Proc Natl Acad Sci U S A 106: 8665–8670.
- 55. Patel A, MacMahon S, Chalmers J, Neal B, Billot L, et al. (2008) Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes. N Engl J Med 358: 2560–2572.