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1.  Relationships between human vitality and mitochondrial respiratory parameters, reactive oxygen species production and dNTP levels in peripheral blood mononuclear cells 
Aging (Albany NY)  2013;5(11):850-864.
Low vitality (a component of fatigue) in middle-aged and older adults is an important complaint often identified as a symptom of a disease state or side effect of a treatment. No studies to date have investigated the potential link between dysfunctional mitochondrial ATP production and low vitality. Therefore, we measured a number of cellular parameters related to mitochondrial activity in peripheral blood mononuclear cells (PBMCs) isolated from middle-aged men, and tested for association with vitality. These parameters estimate mitochondrial respiration, reactive oxygen species (ROS) production, and deoxyribonucleotide (dNTP) balance in PBMCs. The population was drawn from the Metropolit cohort of men born in 1953. Vitality level was estimated from the Medical Outcomes Study Short Form 36 (SF-36) vitality scale. We found that vitality score had no association with any of the mitochondrial respiration parameters. However, vitality score was inversely associated with cellular ROS production and cellular deoxythymidine triphosphate (dTTP) levels and positively associated with deoxycytidine triphosphate (dCTP) levels. We conclude that self-reported persistent low vitality is not associated with specific aspects of mitochondrial oxidative phosphorylation capacity in PBMCs, but may have other underlying cellular dysfunctions that contribute to dNTP imbalance and altered ROS production.
PMCID: PMC3868727  PMID: 24304678
vitality; mitochondrial respiration; reactive oxygen species; deoxyribonucleotides
2.  An Optimized Histochemical Method to Assess Skeletal Muscle Glycogen and Lipid Stores Reveals Two Metabolically Distinct Populations of Type I Muscle Fibers 
PLoS ONE  2013;8(10):e77774.
Skeletal muscle energy metabolism has been a research focus of physiologists for more than a century. Yet, how the use of intramuscular carbohydrate and lipid energy stores are coordinated during different types of exercise remains a subject of debate. Controversy arises from contradicting data from numerous studies, which used different methodological approaches. Here we review the “pros and cons” of previously used histochemical methods and describe an optimized method to ensure the preservation and specificity of detection of both intramyocellular carbohydrate and lipid stores. For optimal preservation of muscle energy stores, air drying cryosections or cycles of freezing-thawing need to be avoided. Furthermore, optimization of the imaging settings in order to specifically image intracellular lipid droplets stained with oil red O or Bodipy-493/503 is shown. When co-staining lipid droplets with associated proteins, Bodipy-493/503 should be the dye of choice, since oil red O creates precipitates on the lipid droplets blocking the light. In order to increase the specificity of glycogen stain, an antibody against glycogen is used. The resulting method reveals the existence of two metabolically distinct myosin heavy chain I expressing fibers: I-1 fibers have a smaller crossectional area, a higher density of lipid droplets, and a tendency to lower glycogen content compared to I-2 fibers. Type I-2 fibers have similar lipid content than IIA. Exhaustive exercise lead to glycogen depletion in type IIA and IIX fibers, a reduction in lipid droplets density in both type I-1 and I-2 fibers, and a decrease in the size of lipid droplets exclusively in type I-1 fibers.
doi:10.1371/journal.pone.0077774
PMCID: PMC3813758  PMID: 24204959
3.  Influence of Erythropoietin on Cognitive Performance during Experimental Hypoglycemia in Patients with Type 1 Diabetes Mellitus: A Randomized Cross-Over Trial 
PLoS ONE  2013;8(4):e59672.
Introduction
The incidence of severe hypoglycemia in type 1 diabetes has not decreased over the past decades. New treatment modalities minimizing the risk of hypoglycemic episodes and attenuating hypoglycemic cognitive dysfunction are needed. We studied if treatment with the neuroprotective hormone erythropoietin (EPO) enhances cognitive function during hypoglycemia.
Materials and Methods
Eleven patients with type 1 diabetes, hypoglycemia unawareness and recurrent severe hypoglycemia completed the study. In a double-blind, randomized, balanced, cross-over study using clamped hypoglycemia they were treated with 40,000 IU of EPO or placebo administered intravenously six days before the two experiments. Cognitive function (primary endpoint), hypoglycemic symptoms, and counter-regulatory hormonal response were recorded.
Results
Compared with placebo, EPO treatment was associated with a significant reduction in errors in the most complex reaction time task (−4.7 (−8.1 to −1.3), p = 0.01) and a less reaction time prolongation (−66 (−117 to −16) msec, p = 0.02). EPO treatment did not change performance in other measures of cognition. Hypoglycemic symptoms, EEG-changes, and counter-regulatory hormone concentrations did not differ between EPO and placebo treatment.
Conclusion
In patients with type 1 diabetes and hypoglycemia unawareness, treatment with EPO is associated with a beneficial effect on cognitive function in a complex reaction time task assessing sustained attention/working memory. Hypoglycemic symptoms and hormonal responses were not changed by EPO treatment.
Trial Registration
ClinicalTrials.gov NCT00615368
doi:10.1371/journal.pone.0059672
PMCID: PMC3618268  PMID: 23577069
4.  Two Weeks of Metformin Treatment Enhances Mitochondrial Respiration in Skeletal Muscle of AMPK Kinase Dead but Not Wild Type Mice 
PLoS ONE  2013;8(1):e53533.
Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5′AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α2 (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.
We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.
doi:10.1371/journal.pone.0053533
PMCID: PMC3544921  PMID: 23341947
5.  Impact of Physical Inactivity on Adipose Tissue Low-Grade Inflammation in First-Degree Relatives of Type 2 Diabetic Patients 
Diabetes Care  2011;34(10):2265-2272.
OBJECTIVE
First-degree relatives (FDRs) of patients with type 2 diabetes may exhibit a disproportionately elevated risk of developing insulin resistance, obesity, and type 2 diabetes when exposed to physical inactivity, which to some unknown extent may involve low-grade inflammation. We investigated whether subjects who are nonobese FDRs show signs of low-grade inflammation before or after exposure to short-term physical inactivity.
RESEARCH DESIGN AND METHODS
We studied 13 healthy FDR subjects and 20 control (CON) subjects matched for age, sex, and BMI before and after 10 days of bed rest (BR). Insulin sensitivity was measured by the hyperinsulinemic euglycemic clamp. Key low-grade inflammation mediators were measured in arterial blood and microdialysate from subcutaneous abdominal (SCAAT) and femoral adipose tissue. Adipokine mRNA expression was determined in SCAAT.
RESULTS
Before BR, FDR subjects displayed insulin resistance, elevated plasma C-reactive protein, leptin, and monocyte chemoattractant protein (MCP)-1, high interleukin (IL)-6, and MCP-1 expressions, as well as low adiponectin and leptin expressions. FDR subjects responded to BR by decreasing plasma adiponectin and IL-10 expression and increasing plasma expression of IL-10 and tumor necrosis factor-α. In contrast, CON subjects responded to BR by increasing plasma adiponectin and adiponectin expression and by decreasing SCAAT microdialysate leptin.
CONCLUSIONS
Young and nonobese FDR of patients with type 2 diabetes exhibit low-grade inflammation, which is further and disproportionately aggravated when exposed to physical inactivity. The study provides support for the notion that people at increased risk of type 2 diabetes should avoid even short periods of physical inactivity.
doi:10.2337/dc11-0631
PMCID: PMC3177724  PMID: 21836102
6.  Insulin resistance and exercise tolerance in heart failure patients: linkage to coronary flow reserve and peripheral vascular function 
Background
Insulin resistance has been linked to exercise intolerance in heart failure patients. The aim of this study was to assess the potential role of coronary flow reserve (CFR), endothelial function and arterial stiffness in explaining this linkage.
Methods
39 patients with LVEF < 35% (median LV ejection fraction (LVEF) 31 (interquartile range (IQ) 26–34), 23/39 of ischemic origin) underwent echocardiography with measurement of CFR. Peak coronary flow velocity (CFV) was measured in the LAD and coronary flow reserve was calculated as the ratio between CFV at rest and during a 2 minutes adenosine infusion. All patients performed a maximal symptom limited exercise test with measurement of peak oxygen uptake (VO2peak), digital measurement of endothelial function and arterial stiffness (augmentation index), dual X-ray absorptiometry scan (DEXA) for body composition and insulin sensitivity by a 2 hr hyperinsulinemic (40 mU/min/m2) isoglycemic clamp.
Results
Fat free mass adjusted insulin sensitivity was significantly correlated to VO2peak (r = 0.43, p = 0.007). Median CFR was 1.77 (IQ 1.26-2.42) and was correlated to insulin sensitivity (r 0.43, p = 0.008). CFR (r = 0.48, p = 0.002), and arterial stiffness (r = −0.35, p = 0.04) were correlated to VO2peak whereas endothelial function and LVEF were not (all p > 0.15). In multivariable linear regression adjusting for age, CFR remained independently associated with VO2peak (standardized coefficient (SC) 1.98, p = 0.05) whereas insulin sensitivity (SC 1.75, p = 0.09) and arterial stiffness (SC −1.17, p = 0.29) were no longer associated with VO2peak.
Conclusions
The study confirms that insulin resistance is associated with exercise intolerance in heart failure patients and suggests that this is partly through reduced CFR. This is the first study to our knowledge that shows an association between CFR and exercise capacity in heart failure patients and links the relationship between insulin resistance and exercise capacity to CFR.
doi:10.1186/1475-2840-11-97
PMCID: PMC3444364  PMID: 22889317
Coronary flow reserve; Heart failure; Exercise capacity; Insulin sensitivity; Arterial stiffness
7.  Erythropoietin Treatment Enhances Muscle Mitochondrial Capacity in Humans 
Erythropoietin (Epo) treatment has been shown to induce mitochondrial biogenesis in cardiac muscle along with enhanced mitochondrial capacity in mice. We hypothesized that recombinant human Epo (rhEpo) treatment enhances skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity in humans. In six healthy volunteers rhEpo was administered by sub-cutaneous injection over 8 weeks with oral iron (100 mg) supplementation taken daily. Mitochondrial OXPHOS was quantified by high-resolution respirometry in saponin-permeabilized muscle fibers obtained from biopsies of the vastus lateralis before and after rhEpo treatment. OXPHOS was determined with the mitochondrial complex I substrates malate, glutamate, pyruvate, and complex II substrate succinate in the presence of saturating ADP concentrations, while maximal electron transport capacity (ETS) was assessed by addition of an uncoupler. rhEpo treatment increased OXPHOS (from 92 ± 5 to 113 ± 7 pmol·s−1·mg−1) and ETS (107 ± 4 to 143 ± 14 pmol·s−1·mg−1, p < 0.05), demonstrating that Epo treatment induces an upregulation of OXPHOS and ETS in human skeletal muscle.
doi:10.3389/fphys.2012.00050
PMCID: PMC3299978  PMID: 22419911
mitochondria; oxidative phosphorylation; rhEPO; muscle; humans
8.  5′-AMP Activated Protein Kinase is Involved in the Regulation of Myocardial β-Oxidative Capacity in Mice 
5′-adenosine monophosphate-activated protein kinase (AMPK) is considered central in regulation of energy status and substrate utilization within cells. In heart failure the energetic state is compromised and substrate metabolism is altered. We hypothesized that this could be linked to changes in AMPK activity and we therefore investigated mitochondrial oxidative phosphorylation capacity from the oxidation of long- and medium-chain fatty acids (LCFA and MCFA) in cardiomyocytes from young and old mice expressing a dominant negative AMPKα2 (AMPKα2-KD) construct and their wildtype (WT) littermates. We found a 35–45% (P < 0.05) lower mitochondrial capacity for oxidizing MCFA in AMPKα2-KD of both age-groups, compared to WT. This coincided with marked decreases in protein expression (19/29%, P < 0.05) and activity (14/21%, P < 0.05) of 3-hydroxyacyl-CoA-dehydrogenase (HAD), in young and old AMPKα2-KD mice, respectively, compared to WT. Maximal LCFA oxidation capacity was similar in AMPKα2-KD and WT mice independently of age implying that LCFA-transport into the mitochondria was unaffected by loss of AMPK activity or progressing age. Expression of regulatory proteins of glycolysis and glycogen breakdown showed equivocal effects of age and genotype. These results illustrate that AMPK is necessary for normal mitochondrial function in the heart and that decreased AMPK activity may lead to an altered energetic state as a consequence of reduced capacity to oxidize MCFA. We did not identify any clear aging effects on mitochondrial function.
doi:10.3389/fphys.2012.00033
PMCID: PMC3284200  PMID: 22371704
AMPK; metabolic remodeling; mitochondria; oxidative phosphorylation
9.  The T-Allele of TCF7L2 rs7903146 Associates With a Reduced Compensation of Insulin Secretion for Insulin Resistance Induced by 9 Days of Bed Rest 
Diabetes  2010;59(4):836-843.
OBJECTIVE
The aim of this study was to determine whether the type 2 diabetes–associated T-allele of transcription factor 7-like 2 (TCF7L2) rs7903146 associates with impaired insulin secretion to compensate for insulin resistance induced by bed rest.
RESEARCH DESIGN AND METHODS
A total of 38 healthy young Caucasian men were studied before and after bed rest using the hyperinsulinemic-euglycemic clamp technique combined with indirect calorimetry preceded by an intravenous glucose tolerance test. The TCF7L2 rs7903146 was genotyped using allelic discrimination performed with an ABI 7900 system. The genetic analyses were done assuming a dominant model of inheritance.
RESULTS
The first-phase insulin response (FPIR) was significantly lower in carriers of the T-allele compared with carriers of the CC genotype before bed rest, with and without correction for insulin resistance. The incremental rise of FPIR in response to insulin resistance induced by bed rest was lower in carriers of the T-allele (P < 0.001). Fasting plasma glucagon levels were significantly lower in carriers of the T-allele before and after bed rest. While carriers of the CC genotype developed increased hepatic insulin resistance, the TCF7L2 rs7903146 did not influence peripheral insulin action or the rate of lipolysis before or after bed rest.
CONCLUSIONS
Healthy carriers of the T-allele of TCF7L2 rs7903146 exhibit a diminished increase of insulin secretion in response to intravenous glucose to compensate for insulin resistance as induced by bed rest. Reduced paracrine glucagon stimulation may contribute to the impairment of β-cell function in the carriers TCF7L2 rs7903146 T-allele associated with increased risk of type 2 diabetes.
doi:10.2337/db09-0918
PMCID: PMC2844831  PMID: 20107109
10.  Impact of 9 Days of Bed Rest on Hepatic and Peripheral Insulin Action, Insulin Secretion, and Whole-Body Lipolysis in Healthy Young Male Offspring of Patients With Type 2 Diabetes 
Diabetes  2009;58(12):2749-2756.
OBJECTIVE
The aim of this study was to investigate the impact of 9 days of bed rest on insulin secretion, insulin action, and whole-body glucose and fat metabolism in first-degree relative (FDR) and matched control (CON) subjects.
RESEARCH DESIGN AND METHODS
A total of 13 FDR and 20 CON subjects participated in the study. All were studied before and after 9 days of bed rest using the clamp technique combined with indirect calorimetry preceded by an intravenous glucose tolerance test. Glucose and glycerol turnover rates were studied using stable isotope kinetics.
RESULTS
Bed rest caused a significant decrease in whole-body insulin sensitivity in both groups. Hepatic insulin resistance was elevated in FDR subjects prior to bed rest and was significantly augmented by bed rest in FDR (P < 0.01) but not in CON (P = NS) subjects. The rate of whole-body lipolysis decreased during bed rest in both FDR and CON subjects, with no significant differences between the groups. Insulin resistance induced by bed rest was fully accounted for by the impairment of nonoxidative glucose metabolism in both groups (overall P < 0.001).
CONCLUSIONS
Whole-body insulin action in both insulin-resistant FDR and healthy CON subjects deteriorates with 9 days of bed rest, converging toward similar degrees of whole-body insulin resistance. FDR subjects exhibit hepatic insulin resistance (HIR), which, in contrast to CON subjects, deteriorates in response to physical inactivity. FDR subjects exhibit reduced insulin secretion when seen in relation to their degree of HIR but not peripheral insulin resistance.
doi:10.2337/db09-0369
PMCID: PMC2780872  PMID: 19720789

Results 1-10 (10)