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Cited by 156Mitochondrial Function and PathologyAdipose Tissue and MetabolismMetabolism, Diabetes, and Cancer

Prolonged fasting identifies skeletal muscle mitochondrial dysfunction as consequence rather than cause of human insulin resistance

Open access
Hoeks, Joris, van Herpen, Noud A., Mensink, Marco, Moonen-Kornips, Esther, van Beurden, Denis, Hesselink, Matthijs K.C., Schrauwen, Patrick·2010·Diabetes
DOI 10.2337/db10-0519PMC PMC2927932
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Abstract

OBJECTIVE: Type 2 diabetes and insulin resistance have been associated with mitochondrial dysfunction, but it is debated whether this is a primary factor in the pathogenesis of the disease. To test the concept that mitochondrial dysfunction is secondary to the development of insulin resistance, we employed the unique model of prolonged fasting in humans. Prolonged fasting is a physiologic condition in which muscular insulin resistance develops in the presence of increased free fatty acid (FFA) levels, increased fat oxidation and low glucose and insulin levels. It is therefore anticipated that skeletal muscle mitochondrial function is maintained to accommodate increased fat oxidation unless factors secondary to insulin resistance exert negative effects on mitochondrial function. RESEARCH DESIGN AND METHODS: While in a respiration chamber, twelve healthy males were subjected to a 60 h fast and a 60 h normal fed condition in a randomized crossover design. Afterward, insulin sensitivity was assessed using a hyperinsulinemic-euglycemic clamp, and mitochondrial function was quantified ex vivo in permeabilized muscle fibers using high-resolution respirometry. RESULTS: Indeed, FFA levels were increased approximately ninefold after 60 h of fasting in healthy male subjects, leading to elevated intramuscular lipid levels and decreased muscular insulin sensitivity. Despite an increase in whole-body fat oxidation, we observed an overall reduction in both coupled state 3 respiration and maximally uncoupled respiration in permeabilized skeletal muscle fibers, which could not be explained by changes in mitochondrial density. CONCLUSIONS: These findings confirm that the insulin-resistant state has secondary negative effects on mitochondrial function. Given the low insulin and glucose levels after prolonged fasting, hyperglycemia and insulin action per se can be excluded as underlying mechanisms, pointing toward elevated plasma FFA and/or intramuscular fat accumulation as possible causes for the observed reduction in mitochondrial capacity.

AI snapshot

Written from the paper’s full text.

TL;DR

In healthy lean men, 60 h of fasting induced insulin resistance together with elevated free fatty acids and intramuscular lipids, and it reduced intrinsic skeletal muscle mitochondrial respiratory capacity without changing mitochondrial density. The authors conclude that mitochondrial dysfunction is a secondary consequence of the insulin-resistant/lipid-exposed state rather than its cause.

Key findings

  • After 60 h fasting, plasma FFA rose approximately ninefold (1,981 ± 95 vs. 387 ± 37 μmol/l, P < 0.001), plasma glucose fell to 3.72 ± 0.13 mmol/l, and plasma insulin fell to 7.0 ± 0.74 μU/ml; insulin sensitivity index decreased by ∼45% (P < 0.001), with clamp glucose Rd falling from 38.0 ± 2.8 to 18.8 ± 0.9 μmol/kg/min.
  • Intramuscular triacylglycerol area fraction increased ∼2.7-fold overall after fasting (P = 0.001), with a ∼3.5-fold increase in type 1 fibers (P < 0.001) and approximately twofold in type 2 fibers (P = 0.015).
  • Mitochondrial density markers were unchanged: mtDNA copy number (P = 0.96), OXPHOS protein levels, and citrate synthase activity (76.2 ± 7.3 vs. 70.2 ± 6.6 μmol/min/g protein, P = 0.41).
  • Despite increased whole-body fat oxidation (24-h RER 0.91 ± 0.009 fed vs. 0.77 ± 0.003 fasted, P < 0.001), mitochondrial respiration was reduced: state 3 on lipid substrate was lower (P = 0.03), state 3 with parallel Complex I+II input was ∼20% lower (MOG P = 0.01; MOGS P = 0.02; MGS P = 0.04), and maximal FCCP-uncoupled respiration was ∼23% lower (P = 0.008); state 4o and UCP3 were unchanged.

Methods

  • Twelve healthy, lean, male volunteers (age 23.6 ± 1.0 years; BMI 22.6 ± 0.5 kg/m²) completed a randomized crossover trial of 60 h fasting versus 60 h normal feeding, with a 2-week washout, while staying in a respiration chamber.
  • After each condition, insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp, and mitochondrial function was measured ex vivo in permeabilized skeletal muscle fibers using high-resolution respirometry with multiple substrate/inhibitor protocols.
  • Additional measures included indirect calorimetry, blood FFA/glucose/insulin, intramuscular triacylglycerol staining, mtDNA copy number, citrate synthase activity, and OXPHOS/UCP3 Western blotting; comparisons used paired Student t tests and repeated-measures ANOVA.

Limitations

  • The paper states that fasting is not a direct model for type 2 diabetes, that it cannot exclude the possibility that reduced mitochondrial function triggers the observed insulin resistance, and that chronic hyperinsulinemia/hyperglycemia may still negatively affect mitochondrial function in type 2 diabetes patients.
  • The paper also notes that the high plasma FFA and IMTG values may reflect complete compliance in the respiration chamber, that species differences complicate comparison with animal high-fat feeding studies, and that it cannot exclude muscle-specific fat oxidation being compensated by other organs or effects on metabolic flexibility.
  • Evident from the text: the sample was small (n = 12), limited to healthy lean men without diabetes family history, and the intervention was short-term (60 h), so causal direction and generalizability to women, obesity, or type 2 diabetes are not established.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.2337/db10-0519

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 45 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    After 60 h of fasting, state 3 respiration upon malate + octanoyl-carnitine + glutamate (MOG) is significantly lower than in the fed condition.

    “Thus, state 3 respiration upon malate + octanoyl-carnitine + glutamate (MOG) was significantly lower in the fasted state compared with the fed condition (P = 0.01, Fig. 5E).”
    Quote in the paperSubstantial claim
  • Findingown data

    After 60 h of fasting, plasma free fatty acid levels increase approximately ninefold in healthy male subjects.

    “Indeed, FFA levels were increased approximately ninefold after 60 h of fasting in healthy male subjects”
    • Also stated in: § Plasma parameters. · plain
      “Upon 60 h of fasting, plasma FFA increased dramatically, up to 1,981 ± 95 μmol/l vs. 387 ± 37 in the fed condition (P < 0.001).”
    Quote in the paperSubstantial claim
  • Findingown data

    The reduction in whole-body insulin sensitivity after 60 h of fasting is mainly accounted for by a reduction in insulin-stimulated glucose disposal.

    “The reduction in whole-body insulin sensitivity was mainly accounted for by a reduction in insulin-stimulated glucose disposal (ΔRd, P < 0.001, Table 2).”
    Quote in the paperSubstantial claim
  • Findingown data

    After 60 h of fasting, citrate synthase activity is similar to the fed condition.

    “and CS activity (76.2 ± 7.3 vs. 70.2 ± 6.6 μmol/min/g protein, P = 0.41) were similar in the fed versus the fasted state, respectively, confirming an equal mitochondrial mass in both conditions.”
    Quote in the paperSubstantial claim
  • Findingown data

    After 60 h of fasting, state 3 respiration upon malate + glutamate + succinate (MGS) is significantly lower than in the fed condition.

    “Similar differences were observed for state 3 respiration upon malate + octanoyl-carnitine + glutamate + succinate (MOGS, P = 0.02, Fig. 5E) and malate + glutamate + succinate (MGS, P = 0.04, Fig. 5E).”
    Quote in the paperSubstantial claim
  • May combine two claims. 22
  • Classified as probably not a substantial claim. 30

  • May combine two claims.

    Findingown data

    After 60 h of fasting, state 3 respiration upon Complex I substrates malate + glutamate is approximately 22% lower, but this difference is not statistically significant.

    “Similarly, state 3 upon the Complex I substrates malate + glutamate (MG) was ∼22% lower after fasting, although this did not reach statistical significance (P = 0.12, Fig. 5D).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    After 60 h of fasting, plasma glucose levels decrease in healthy male subjects.

    “During fasting, however, plasma glucose levels gradually decreased to 3.72 ± 0.13 mmol/l at t = 60 h (P < 0.001).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    mtDNA copy number, CS activity, and OXPHOS protein levels remained unaffected by fasting.

    “mtDNA copy number, CS activity, and OXPHOS protein levels remained unaffected by fasting.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    The absolute plasma FFA values achieved in this study (~2.0 mol/l) are high.

    “the absolute values achieved in this study (∼2.0 mol/l) are high.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    Plasma FFA increased upon prolonged fasting.

    “The impressive increase in plasma FFA upon prolonged fasting is in line with previous findings in humans (12,39), although the absolute values achieved in this study (∼2.0 mol/l) are high.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    The ~2.7-fold increase in IMTG levels after 60 h of fasting is slightly higher in comparison with previous reports.

    “the ∼2.7-fold increase in IMTG levels after 60 h of fasting is slightly higher in comparison with previous reports (12,39).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [12] Stannard, 2002, Fasting for 72 h increases intramyocellular lipid content in nondiabetic, physically fit men, Am J Physiol Endocrinol Metab source

      Not checked in the free run.

    • cites [39] Johnson, 2006, Effect of short-term starvation versus high-fat diet on intramyocellular triglyceride accumulation and insulin resistance in physically fit men, Exp Physiol source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    60 h of fasting in humans lowered insulin-stimulated glucose uptake down to ~50% along with drastically elevated plasma FFA and IMTG levels.

    “60 h of fasting in humans lowered insulin-stimulated glucose uptake down to ∼50% along with drastically elevated plasma FFA and IMTG levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The reduction in insulin-stimulated glucose uptake observed in the present study confirms previous observations showing that prolonged fasting reduced glucose Rd, which was accounted for by a reduction in both insulin-stimulated glucose oxidation and nonoxidative glucose disposal.

    “The reduction in insulin-stimulated glucose uptake observed in the present study confirms previous observations showing that prolonged fasting reduced glucose Rd, which was accounted for by a reduction in both insulin-stimulated glucose oxidation and nonoxidative glucose disposal (40).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Bergman, 2007, Effects of fasting on insulin action and glucose kinetics in lean and obese men and women, Am J Physiol Endocrinol Metab source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The reduction in insulin-stimulated glucose uptake was accompanied by an overall reduction in intrinsic mitochondrial functional capacity in skeletal muscle, despite a pronounced increase in whole-body fat oxidation.

    “This was accompanied by an overall reduction in intrinsic mitochondrial functional capacity in skeletal muscle, despite a pronounced increase in whole-body fat oxidation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Insulin can stimulate mitochondrial biogenesis and increase ATP synthesis in skeletal muscle.

    “it has been shown that insulin can stimulate mitochondrial biogenesis and increases ATP synthesis in skeletal muscle (6,7).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [6] Pagel-Langenickel, 2008, PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle, J Biol Chem source

      Not checked in the free run.

    • cites [7] Asmann, 2006, Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia, Diabetes source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Short-term elevation of lipid availability reduces insulin-stimulated increase in ATP synthase flux in skeletal muscle.

    “Moreover, it was shown in a comparable study that short-term elevation of lipid availability reduces insulin-stimulated increase in ATP synthase flux in skeletal muscle (10), although this may mainly reflect an effect of muscular insulin resistance on ATP flux.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Brehm, 2006, Increased lipid availability impairs insulin-stimulated ATP synthesis in human skeletal muscle, Diabetes source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Plasma FFA levels negatively correlate with mitochondrial function measured by magnetic resonance spectroscopy.

    “Thus, Szendroedi et al. (8) showed that plasma FFA levels negatively correlated with mitochondrial function measured by magnetic resonance spectroscopy.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Szendroedi et al. (8) Szendroedi, 2007, Muscle mitochondrial ATP synthesis and glucose transport/phosphorylation in type 2 diabetes, PLoS Med source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Elevated plasma FFA and/or intramuscular fat accumulation are possible causes for the observed reduction in mitochondrial capacity after prolonged fasting.

    “pointing toward elevated plasma FFA and/or intramuscular fat accumulation as possible causes for the observed reduction in mitochondrial capacity.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Ex vivo high-resolution respirometry in permeabilized skeletal muscle fibers reflects in vivo skeletal muscle mitochondrial functional capacity.
    • Rests on an assumption: The prolonged fasting model in healthy males is a valid model for testing whether mitochondrial dysfunction is secondary to insulin resistance in humans.

  • Classified as probably not a substantial claim.

    Premisecitation

    Exposing human primary muscle cells to insulin upregulates the expression of PGC-1α.

    “it has been reported that exposing human primary muscle cells to insulin upregulates the expression of PGC-1α (27).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [27] Al-Khalili, 2005, Enhanced insulin-stimulated glycogen synthesis in response to insulin, metformin or rosiglitazone is associated with increased mRNA expression of GLUT4 and peroxisomal proliferator activator receptor gamma co-activator 1, Diabetologia source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Subjects stayed in a respiration chamber during the entire 60 h to ensure compliance to the dietary regime.

    “Subject stayed in a respiration chamber during the entire 60 h to ensure compliance to the dietary regime and to allow the measurement of 24 h substrate oxidation and energy expenditure (16).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Several approaches to elevate plasma FFA levels are accompanied by hyperinsulinemia and/or hyperglycemia and insulin resistance, all factors that have also been suggested to interfere with mitochondrial capacity.

    “several approaches (high-fat feeding and lipid infusion combined with a hyperinsulinemic-euglycemic clamp) to elevate plasma FFA levels are accompanied by hyperinsulinemia and/or hyperglycemia and insulin resistance, all factors that have also been suggested to interfere with mitochondrial capacity (26,29).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [26] Stump, 2003, Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts, Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [29] Yu, 2006, Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology, Proc Natl Acad Sci U S A source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Fasting has been quite convincingly shown to increase UCP3 protein levels in animal studies.

    “fasting has been quite convincingly shown to increase UCP3 protein levels in animal studies (36,37).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [36] Moreno, 2003, Fasting, lipid metabolism, and triiodothyronine in rat gastrocnemius muscle: interrelated roles of uncoupling protein 3, mitochondrial thioesterase, and coenzyme Q, FASEB J source

      Not checked in the free run.

    • cites [37] Cadenas, 1999, UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged, FEBS Lett source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The study protocol was approved by the Medical Ethical Committee of Maastricht University Medical Centre and all subjects gave written informed consent.

    “The study protocol was reviewed and approved by the Medical Ethical Committee of Maastricht University Medical Centre and all subjects gave their written informed consent before participating in the study.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Mitochondrial membrane potential was impaired upon short-term lipid infusion in healthy individuals, although several other markers of mitochondrial function remained unaffected.

    “mitochondrial membrane potential was impaired upon short-term lipid infusion in healthy individuals, although several other markers of mitochondrial function remained unaffected (32).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Chavez, 2009, Effect of short-term free fatty acids elevation on mitochondrial function in skeletal muscle of healthy individuals, J Clin Endocrinol Metab

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    High-fat feeding for 8 weeks in rats resulted in a twofold increase of PGC-1α protein levels.

    “high-fat feeding for 8 weeks in rats resulted in a twofold increase of PGC-1α protein levels (34).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Hoeks, 2008, Mitochondrial function, content and ROS production in rat skeletal muscle: effect of high-fat feeding, FEBS Lett source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    PGC-1α expression and the insulin-stimulated increase in skeletal muscle ATP synthesis were reduced upon lipid infusion.

    “PGC-1α expression (9) and the insulin-stimulated increase in skeletal muscle ATP synthesis (10) were reduced upon lipid infusion.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [9] Hoeks, 2006, Peroxisome proliferator-activated receptor-gamma coactivator-1 and insulin resistance: acute effect of fatty acids, Diabetologia source

      Not checked in the free run.

    • cites [10] Brehm, 2006, Increased lipid availability impairs insulin-stimulated ATP synthesis in human skeletal muscle, Diabetes source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    The average increase in oxygen consumption upon cytochrome C was less than 10% and similar in both conditions.

    “The average increase in oxygen consumption upon cytochrome C was less than 10% (underscoring the viability of the muscle fibers) and similar in both conditions (P = 0.54).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In mice, prolonged consumption of a high-fat diet for 16 weeks reduced mitochondrial function.

    “it was shown in mice that prolonged consumption of a high-fat diet for the duration of 16 weeks reduced mitochondrial function (31).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] Bonnard, 2008, Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice, J Clin Invest

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All measurements were performed in duplicate.

    “All measurements were performed in duplicate.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    At low doses of insulin (reflecting postabsorptive levels), the skeletal mitochondrial ATP synthesis rate was not different between diabetic patients and controls, indicating that there is no intrinsic muscle mitochondrial defect in type 2 diabetic patients.

    “it was demonstrated that at low doses of insulin (reflecting postabsorptive levels) the skeletal mitochondrial ATP synthesis rate was not different between diabetic patients and controls, indicating that there is no intrinsic muscle mitochondrial defect in type 2 diabetic patients (7).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7] Asmann, 2006, Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia, Diabetes source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Severe hyperglycemia inhibited respiration in human skeletal muscle, which was restored upon insulin treatment.

    “it was reported that severe hyperglycemia inhibited respiration in human skeletal muscle, which was restored upon insulin treatment (13).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [13] Rabol, 2009, Effect of hyperglycemia on mitochondrial respiration in type 2 diabetes, J Clin Endocrinol Metab source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The increase in plasma FFA upon prolonged fasting is in line with previous findings in humans.

    “The impressive increase in plasma FFA upon prolonged fasting is in line with previous findings in humans (12,39)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [12] Stannard, 2002, Fasting for 72 h increases intramyocellular lipid content in nondiabetic, physically fit men, Am J Physiol Endocrinol Metab source

      Not checked in the free run.

    • cites [39] Johnson, 2006, Effect of short-term starvation versus high-fat diet on intramyocellular triglyceride accumulation and insulin resistance in physically fit men, Exp Physiol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    An alternative candidate to explain the observed reduction in mitochondrial capacity upon fasting is prolonged exposure to elevated plasma FFA levels.

    “An alternative candidate to explain the observed reduction in mitochondrial capacity upon fasting is prolonged exposure to elevated plasma FFA levels.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Hyperglycemia increases mitochondrial ROS production in endothelial cells, as well as in other cell types.

    “hyperglycemia has been shown to increase mitochondrial ROS production in endothelial cells (28), as well as in other cell types (29).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [28] Brownlee, 2005, The pathobiology of diabetic complications: a unifying mechanism, Diabetes source

      Not checked in the free run.

    • cites [29] Yu, 2006, Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology, Proc Natl Acad Sci U S A source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Oxygen fluxes were adjusted for individual differences in mtDNA copy number, and similar results were obtained without this correction.

    “Nonetheless, we adjusted the oxygen fluxes for individual differences in mtDNA copy number. However, similar results were obtained without this correction (see supplemental Fig. 1 in the online appendix available at http://diabetes.diabetesjournals.org).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    In healthy individuals, a 7 h insulin infusion increased mitochondrial protein synthesis, cytochrome C oxidase (COX), and citrate synthase (CS) enzyme activities and ATP production.

    “in healthy individuals, it was shown that a 7 h insulin infusion increased mitochondrial protein synthesis, cytochrome C oxidase (COX), and citrate synthase (CS) enzyme activities and ATP production (26).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Stump, 2003, Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts, Proc Natl Acad Sci U S A source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationnone given

    Fasting is not a direct model for type 2 diabetes.

    “However, it should be noted that fasting is not a direct model for type 2 diabetes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Differences in absolute levels of plasma FFA achieved in the different studies may contribute to (part of) the variation.

    “differences in absolute levels of plasma FFA achieved in the different studies may contribute to (part of) the variation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    The study used a randomized crossover design with a 2-week washout period.

    “Subjects participated in two experimental trials: a 60 h fast and a 60 h normal fed condition, in a randomized crossover design with a 2-week washout period.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    The available data in the literature underpin the notion that fasting-induced lipid accumulation is responsible for reduced insulin sensitivity upon fasting.

    “although the available data in the literature underpin the notion that fasting-induced lipid accumulation is responsible for reduced insulin sensitivity upon fasting”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    UCP3 mRNA levels were elevated after 15 h (~5-fold) and 40 h (~10-fold) of fasting in humans.

    “UCP3 mRNA levels were also elevated after 15 h (∼5-fold) and 40 h (∼10-fold) of fasting in humans (38).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Tunstall, 2002, Fasting activates the gene expression of UCP3 independent of genes necessary for lipid transport and oxidation in skeletal muscle, Biochem Biophys Res Commun source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Both a reduction in the activity of the electron transport chain and the oxidative phosphorylation system could underlie the reduced state 3 respiration.

    “Therefore, both a reduction in the activity of the electron transport chain and the oxidative phosphorylation system could underlie the reduced state 3 respiration.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Raising plasma FFA by high-fat feeding combined with daily heparin injections for 4 weeks in rats increased skeletal muscle mitochondrial biogenesis and mitochondrial enzymes involved in fat oxidation, the citric acid cycle, and the respiratory chain.

    “raising plasma FFA by high-fat feeding combined with daily heparin injections for 4 weeks in rats increased skeletal muscle mitochondrial biogenesis and mitochondrial enzymes involved in fat oxidation, the citric acid cycle, and the respiratory chain (33).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] Garcia-Roves, 2007, Raising plasma fatty acid concentration induces increased biogenesis of mitochondria in skeletal muscle, Proc Natl Acad Sci U S A source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    High (postprandial) levels of insulin increased the mitochondrial ATP production rate in nondiabetic subjects, whereas this increase was absent in type 2 diabetic patients.

    “high (postprandial) levels of insulin increased the mitochondrial ATP production rate in nondiabetic subjects, whereas this increase was absent in type 2 diabetic patients (7).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7] Asmann, 2006, Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia, Diabetes source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The decrease in mitochondrial capacity in skeletal muscle is counterintuitive, especially since this decrease was substrate-independent and also apparent upon a lipid substrate.

    “Therefore, the decrease in mitochondrial capacity in skeletal muscle is counterintuitive, especially since this decrease was substrate-independent and also apparent upon a lipid substrate.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The high plasma FFA and IMTG levels might be caused by complete compliance to the fasting regimen in the present study since, in contrast to other studies, the subjects stayed in a respiration chamber throughout the whole period.

    “The high plasma FFA and IMTG levels might be caused by complete compliance to the fasting regimen in the present study since, in contrast to other studies, the subjects stayed in a respiration chamber throughout the whole period.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    It has generally been assumed that a reduction in muscle mitochondrial function will result in reductions in whole-body fat oxidative capacity.

    “it has generally been assumed that a reduction in muscle mitochondrial function will result in reductions in whole-body fat oxidative capacity (3–5,42,43).”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [3] Kelley, 2002, Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes, Diabetes source

      Not checked in the free run.

    • cites [4] Phielix, 2008, Lower intrinsic ADP-stimulated mitochondrial respiration underlies in vivo mitochondrial dysfunction in muscle of male type 2 diabetic patients, Diabetes source

      Not checked in the free run.

    • cites [5] Mogensen, 2007, Mitochondrial respiration is decreased in skeletal muscle of patients with type 2 diabetes, Diabetes source

      Not checked in the free run.

    • cites [42] Ortenblad, 2005, Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes: evidence for an intrinsic oxidative enzyme defect, Biochim Biophys Acta source

      Not checked in the free run.

    • cites [43] Ritov, 2005, Deficiency of subsarcolemmal mitochondria in obesity and type 2 diabetes, Diabetes source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Prolonged fasting reduced glucose Rd, which was accounted for by a reduction in both insulin-stimulated glucose oxidation and nonoxidative glucose disposal.

    “previous observations showing that prolonged fasting reduced glucose Rd, which was accounted for by a reduction in both insulin-stimulated glucose oxidation and nonoxidative glucose disposal (40).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Bergman, 2007, Effects of fasting on insulin action and glucose kinetics in lean and obese men and women, Am J Physiol Endocrinol Metab source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Not all studies show the effect of prolonged fasting on metabolic flexibility.

    “However, not all studies show this effect (41).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Soeters, 2008, Muscle adaptation to short-term fasting in healthy lean humans, J Clin Endocrinol Metab source

      Not checked in the free run.

44 of 44

  • The paper relies on this without support.

    Ex vivo high-resolution respirometry in permeabilized skeletal muscle fibers reflects in vivo skeletal muscle mitochondrial functional capacity.

    “mitochondrial function was quantified ex vivo in permeabilized muscle fibers using high-resolution respirometry.”

  • The paper relies on this without support.

    The prolonged fasting model in healthy males is a valid model for testing whether mitochondrial dysfunction is secondary to insulin resistance in humans.

    “To test the concept that mitochondrial dysfunction is secondary to the development of insulin resistance, we employed the unique model of prolonged fasting in humans.”

  • The paper relies on this without support.

    Prolonged fasting in humans is a valid model of the insulin-resistant state for determining whether mitochondrial dysfunction is a consequence rather than a cause of insulin resistance.

    “the insulin-resistant state after prolonged fasting”
    “However, it should be noted that fasting is not a direct model for type 2 diabetes.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Fasted nonoxidative glucose-disposal change does not match its table values

    Minor

    Table 2 reports fasted basal and clamp nonoxidative glucose disposal of 4.2 and 12.6 μmol/kg/min. Their difference is 8.4, not the reported delta of 8.2.

    Reported 8.2 · recomputed 8.4

  • Fasted clamp glucose-disposal components do not sum to total disposal

    Minor

    Table 2 gives fasted clamp carbohydrate oxidation as 6.6 and nonoxidative glucose disposal as 12.6 μmol/kg/min. These sum to 19.2, rather than the reported glucose Rd of 18.8; the corresponding fed values sum correctly.

    Reported 18.8 · recomputed 19.2

  • Plasma FFA concentration is stated in incompatible units

    Minor

    The discussion calls the achieved plasma FFA concentration approximately 2.0 mol/l, whereas the results give approximately 1,981 μmol/l. The latter is approximately 2.0 mmol/l, a thousandfold lower than 2.0 mol/l.

3 of 3

Cites

Flagged first, then by citations

1 of the 44 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Hoeks, J., van Herpen, N. A., Mensink, M., Moonen-Kornips, E., van Beurden, D., Hesselink, M. K. C., & Schrauwen, P.
Affiliations
  • School for Nutrition, Toxicology and Metabolism, Department of Human Biology, Maastricht University Medical Centre, Maastricht, the Netherlands;
  • Wageningen University & Research (NL)
  • Maastricht University Medical Centre (NL)
ORCID
Venue
Diabetes
Publisher
American Diabetes Association
Published
2010-06-23
Volume
59
Issue
9
Pages
2117-2125
ISSN
  • 0012-1797 (ISSN-L)
  • 1939-327X
DOI
10.2337/db10-0519
PMC
PMC2927932
OpenAlex
W2134172979
Semantic Scholar
44db7ec23aadf67e5033f75f26c8e2276e3c7411
Cited by
156
References
44
Citations by year
2026: 4 · 2025: 4 · 2024: 2 · 2023: 15 · 2022: 5 · 2021: 9 · 2020: 9 · 2019: 5 · 2018: 11 · 2017: 10 · 2016: 10 · 2015: 7 · 2014: 15 · 2013: 19 · 2012: 15
FWCI
6.51
Type
journal article
Language
en
Open access
Licence
Locations
  • Diabetes (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Research Publications (Maastricht University) (acceptedVersion, cc-by-nc-nd)
  • Socio-Environmental Systems Modeling (acceptedVersion)
  • Research Publications (Maastricht University) (submittedVersion, other-oa)
  • Data Archiving and Networked Services (DANS) (submittedVersion)
Funders
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (918.96.618, 917.66.359)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Health Sciences › Medicine › Physiology › Adipose Tissue and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
Keywords
prolonged fasting, insulin resistance, free fatty acids, fat oxidation, type 2 diabetes pathogenesis, mitochondrial respiration, hyperinsulinemic-euglycemic clamp, high-resolution respirometry, skinned muscle fibers
MeSH terms
Adult, Body Weight, Citrate (si)-Synthase, Diabetes Mellitus, Type 2, DNA, Mitochondrial, Fasting, Fatty Acids, Nonesterified, Humans, Hyperinsulinism, Insulin, Insulin Resistance, Lipids, Male, Mitochondria, Reference Values, Triglycerides, Glucose Clamp Technique, Muscle, Skeletal, Young Adult

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

Statistics and arithmetic are recomputed, not judged.

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