Leslie A. Consitt

Effects of acute aerobic and anaerobic exercise on blood markers of oxidative stress

The purpose of this study was to compare oxidative modification of blood proteins, lipids, DNA, and glutathione in the 24 hours following aerobic and anaerobic exercise using similar muscle groups. Ten cross-trained men (24.3 ± 3.8 years, [mean ± SEM]) performed in random order 30 minutes of continuous cycling at 70% of VO2max and intermittent dumbbell squatting at 70% of 1 repetition maximum (1RM), separated by 1–2 weeks, in a crossover design. Blood samples taken before, and immediately, 1, 6, and 24 hours postexercise were analyzed for plasma protein carbonyls (PC), plasma malondialdehyde (MDA), and whole-blood total (TGSH), oxidized (GSSG), and reduced (GSH) glutathione. Blood samples taken before and 24 hours postexercise were analyzed for serum 8-hydroxy-29-deoxyguanosine (8-OHdG). PC values were greater at 6 and 24 hours postexercise compared with pre-exercise for squatting, with greater PC values at 24 hours postexercise for squatting compared with cycling (0.634 ± 0.053 vs. 0.359 ± 0.018 nM·mg protein-1). There was no significant interaction or main effects for MDA or 8-OHdG. GSSG experienced a shortlived increase and GSH a transient decrease immediately following both exercise modes. These data suggest that 30 minutes of aerobic and anaerobic exercise performed by young, cross-trained men (a) can increase certain biomarkers of oxidative stress in blood, (b) differentially affect oxidative stress biomarkers, and (c) result in a different magnitude of oxidation based on the macromolecule studied. Practical applications: While protein and glutathione oxidation was increased following acute exercise as performed in this study, future research may investigate methods of reducing macromolecule oxidation, possibly through the use of antioxidant therapy.

Intramuscular Lipid Metabolism, Insulin Action and Obesity

With the increasing prevalence of obesity, research has focused on the molecular mechanism(s) linking obesity and skeletal muscle insulin resistance. Metabolic alterations within muscle, such as changes in the cellular location of fatty acid transporter proteins, decreased mitochondrial enzyme activity, and defects in mitochondrial morphology, likely contribute to obesity and insulin resistance. These defects are thought to play a role in the reduced skeletal muscle fatty acid oxidation and increased intramuscular lipid (IMCL) accumulation that is apparent with obesity and other insulin‐resistant states such as type 2 diabetes. Intramuscular triacylglycerol does not appear to be a ubiquitous marker of insulin resistance, although specific IMCL intermediates such as long‐chain fatty acyl‐CoAs, ceramide, and diacylglycerol may inhibit insulin signal transduction. In this review, we will briefly summarize the defects in skeletal muscle lipid metabolism associated with obesity, and discuss the proposed mechanisms by which these defects may contribute to insulin resistance.

Lipid Partitioning, Incomplete Fatty Acid Oxidation, and Insulin Signal Transduction in Primary Human Muscle Cells: Effects of Severe Obesity, Fatty Acid Incubation, and Fatty Acid Translocase/CD36 Overexpression

CONTEXT Intracellular lipid partitioning toward storage and the incomplete oxidation of fatty acids (FA) have been linked to insulin resistance. OBJECTIVE To gain insight into how intracellular lipid metabolism is related to insulin signal transduction, we examined the effects of severe obesity, excess FA, and overexpression of the FA transporter, FA translocase (FAT)/CD36, in primary human skeletal myocytes. DESIGN, SETTING, AND PATIENTS Insulin signal transduction, FA oxidation, and metabolism were measured in skeletal muscle cells harvested from lean and severely obese women. To emulate the obesity phenotype in our cell culture system, we incubated cells from lean individuals with excess FA or overexpressed FAT/CD36 using recombinant adenoviral technology. RESULTS Complete oxidation of FA was significantly reduced, whereas total lipid accumulation, FA esterification into lipid intermediates, and incomplete oxidation were up-regulated in the muscle cells of severely obese subjects. Insulin signal transduction was reduced in the muscle cells from severely obese subjects compared to lean controls. Incubation of muscle cells from lean subjects with lipids reduced insulin signal transduction and increased lipid storage and incomplete FA oxidation. CD36 overexpression increased FA transport capacity, but did not impair complete FA oxidation and insulin signal transduction in muscle cells from lean subjects. CONCLUSIONS Cultured myocytes from severely obese women express perturbations in FA metabolism and insulin signaling reminiscent of those observed in vivo. The obesity phenotype can be recapitulated in muscle cells from lean subjects via exposure to excess lipid, but not by overexpressing the FAT/CD36 FA transporter.

A high-fat diet elicits differential responses in genes coordinating oxidative metabolism in skeletal muscle of lean and obese individuals.

CONTEXT In lean individuals, increasing dietary lipid can elicit an increase in whole body lipid oxidation; however, with obesity the capacity to respond to changes in substrate availability appears to be compromised. OBJECTIVE To determine whether the responses of genes regulating lipid oxidation in skeletal muscle differed between lean and insulin resistant obese humans upon exposure to a high-fat diet (HFD). DESIGN AND SETTING A 5-d prospective study conducted in the research unit of an academic center. PARTICIPANTS Healthy, lean (n = 12; body mass index = 22.1 ± 0.6 kg/m(2)), and obese (n=10; body mass index = 39.6 ± 1.7 kg/m(2)) males and females, between ages 18 and 30. INTERVENTION Participants were studied before and after a 5-d HFD (65% fat). MAIN OUTCOME MEASURES Skeletal muscle biopsies (vastus lateralis) were obtained in the fasted and fed states before and after the HFD and mRNA content for genes involved with lipid oxidation determined. Skeletal muscle acylcarnitine content was determined in the fed states before and after the HFD. RESULTS Peroxisome proliferator activated receptor (PPAR) α mRNA content increased in lean, but not obese, subjects after a single high-fat meal. From Pre- to Post-HFD, mRNA content exhibited a body size × HFD interaction, where the lean individuals increased while the obese individuals decreased mRNA content for pyruvate dehydrogenase kinase 4, uncoupling protein 3, PPARα, and PPARγ coactivator-1α (P ≤ 0.05). In the obese subjects medium-chain acylcarnitine species tended to accumulate, whereas no change or a reduction was evident in the lean individuals. CONCLUSIONS These findings indicate a differential response to a lipid stimulus in the skeletal muscle of lean and insulin resistant obese humans.

Peroxisome Proliferator–Activated Receptor-γ Coactivator-1α Overexpression Increases Lipid Oxidation in Myocytes From Extremely Obese Individuals

OBJECTIVE To determine whether the obesity-related decrement in fatty acid oxidation (FAO) in primary human skeletal muscle cells (HSkMC) is linked with lower mitochondrial content and whether this deficit could be corrected via overexpression of peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α). RESEARCH DESIGN AND METHODS FAO was studied in HSkMC from lean (BMI 22.4 ± 0.9 kg/m2; N = 12) and extremely obese (45.3 ± 1.4 kg/m2; N = 9) subjects. Recombinant adenovirus was used to increase HSkMC PGC-1α expression (3.5- and 8.0-fold), followed by assessment of mitochondrial content (mtDNA and cytochrome C oxidase IV [COXIV]), complete (14CO2 production from labeled oleate), and incomplete (acid soluble metabolites [ASM]) FAO, and glycerolipid synthesis. RESULTS Obesity was associated with a 30% decrease (P < 0.05) in complete FAO, which was accompanied by higher relative rates of incomplete FAO ([14C]ASM production/14CO2), increased partitioning of fatty acid toward storage, and lower (P < 0.05) mtDNA (−27%), COXIV (−35%), and mitochondrial transcription factor (mtTFA) (−43%) protein levels. PGC-1α overexpression increased (P < 0.05) FAO, mtDNA, COXIV, mtTFA, and fatty acid incorporation into triacylglycerol in both lean and obese groups. Perturbations in FAO, triacylglycerol synthesis, mtDNA, COXIV, and mtTFA in obese compared with lean HSkMC persisted despite PGC-1α overexpression. When adjusted for mtDNA and COXIV content, FAO was equivalent between lean and obese groups. CONCLUSION Reduced mitochondrial content is related to impaired FAO in HSkMC derived from obese individuals. Increasing PGC-1α protein levels did not correct the obesity-related absolute reduction in FAO or mtDNA content, implicating mechanisms other than PGC-1α abundance.

Impairments in site-specific AS160 phosphorylation and effects of exercise training

The purpose of this study was to determine if site-specific phosphorylation at the level of Akt substrate of 160 kDa (AS160) is altered in skeletal muscle from sedentary humans across a wide range of the adult life span (18–84 years of age) and if endurance- and/or strength-oriented exercise training could rescue decrements in insulin action and skeletal muscle AS160 phosphorylation. A euglycemic-hyperinsulinemic clamp and skeletal muscle biopsies were performed in 73 individuals encompassing a wide age range (18–84 years of age), and insulin-stimulated AS160 phosphorylation was determined. Decrements in whole-body insulin action were associated with impairments in insulin-induced phosphorylation of skeletal muscle AS160 on sites Ser-588, Thr-642, Ser-666, and phospho-Akt substrate, but not Ser-318 or Ser-751. Twelve weeks of endurance- or strength-oriented exercise training increased whole-body insulin action and reversed impairments in AS160 phosphorylation evident in insulin-resistant aged individuals. These findings suggest that a dampening of insulin-induced phosphorylation of AS160 on specific sites in skeletal muscle contributes to the insulin resistance evident in a sedentary aging population and that exercise training is an effective intervention for treating these impairments.

Phosphorylation of the JAK2–STAT5 Pathway in Response to Acute Aerobic Exercise

UNLABELLED Growth hormone (GH) is a powerful stimulator of the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) pathway. Acute exercise is a known stimulus for GH secretion. PURPOSE The purpose of this study was to determine the phosphorylation of the JAK2-STAT5 pathway in human skeletal muscle in response to acute aerobic exercise. METHODS Eleven young (22.5 +/- 0.6, mean +/- SE), healthy, aerobically trained males performed 30 min of cycling at 70% V O2max. Blood samples were collected at 10- to 15-min intervals and analyzed for human GH, immunofunctional (IF) GH, GH binding protein, and insulin-like growth factor I (IGF-I). Muscle biopsies were taken from the vastus lateralis before exercise, immediately after exercise, as well as, 30 and 60 min postexercise. Muscle samples were analyzed for changes in JAK2 and STAT5 tyrosine phosphorylation, as well as changes in JAK2 and STAT5 protein content. RESULTS Multivariate ANOVA with post hoc comparisons demonstrated that GH and IF GH were significantly elevated immediately after exercise compared with preexercise (P < 0.001). Exercise significantly increased the phosphorylation of JAK2 immediately after exercise (P = 0.004). A trend toward increasing levels of STAT5 phosphorylation was observed immediately after exercise (P = 0.08) and was significantly elevated 30 min after exercise (P = 0.002), compared with preexercise levels. Muscle JAK2 and STAT5 protein content did not change. CONCLUSION The results demonstrate that the JAK2-STAT5 pathway is activated in response to acute aerobic exercise in human skeletal muscle and suggests that the exercise-induced release of GH may play a role in the activation of this pathway.

Age-related impairments in skeletal muscle PDH phosphorylation and plasma lactate are indicative of metabolic inflexibility and the effects of exercise training

The purpose of this study was to determine whether plasma lactate and skeletal muscle glucose regulatory pathways, specifically PDH dephosphorylation, are impaired during hyperinsulinemic conditions in middle- to older-aged individuals and determine whether exercise training could improve key variables responsible for skeletal muscle PDH regulation. Eighteen young (19-29 yr; n = 9 males and 9 females) and 20 middle- to older-aged (57-82 yr; n = 10 males and 10 females) individuals underwent a 2-h euglycemic hyperinsulinemic clamp. Plasma samples were obtained at baseline and at 30, 50, 90, and 120 min for analysis of lactate, and skeletal muscle biopsies were performed at 60 min for analysis of protein associated with glucose metabolism. In response to insulin, plasma lactate was elevated in aged individuals when normalized to insulin action. Insulin-stimulated phosphorylation of skeletal muscle PDH on serine sites 232, 293, and 300 decreased in young individuals only. Changes in insulin-stimulated PDH phosphorylation were positively related to changes in plasma lactate. No age-related differences were observed in skeletal muscle phosphorylation of LDH, GSK-3α, or GSK-3β in response to insulin or PDP1, PDP2, PDK2, PDK4, or MPC1 total protein. Twelve weeks of endurance- or strength-oriented exercise training improved insulin-stimulated PDH dephosphorylation, which was related to a reduced lactate response. These findings suggest that impairments in insulin-induced PDH regulation in a sedentary aging population contribute to impaired glucose metabolism and that exercise training is an effective intervention for treating metabolic inflexibility.

Substrate utilization during and after exercise in mild cystic fibrosis.

PURPOSE To determine substrate utilization and energy expenditure during maximal and submaximal exercise and recovery in adolescents with cystic fibrosis (CF) and healthy age-matched controls (C). METHODS Ten clinically stable CF patients (four girls, six boys; age = 10-22 yr) were matched by body mass index, age, gender, and Tanner stage to healthy controls. Subjects completed VO(2peak) testing and submaximal exercise (20 min) on a cycle ergometer at a relative intensity of 50% VO2(peak) and at an absolute power output (PO). Metabolic parameters were assessed during exercise and recovery (20 min). RESULTS Similar respiratory quotient (RQ) values occurred in both groups during maximal exercise and recovery, despite differences in the maximal PO [CF = 114 (60-180) W and C = 171 (105-280) W, P = 0.006], the total work completed [CF = 27 (9.0-54.3) kJ and C = 55 (25.3-126.5) kJ, P = 0.008], or the VO(2peak) value attained [CF = 30.6 (8.5-45.2) mL kg(-1) min(-1) and C = 40.6 (29-64.5) mL kg(-1) min(-1), P = 0.027]. Submaximal exercise at the same absolute PO resulted in similar RQ values during exercise and recovery despite higher heart rates and average VO2 [CF = 18.8 (9.3-28.7) mL kg(-1) min(-1) and C = 15.2 (6.5-20.1) mL kg(-1)min(-1), P = 0.031] values in CF adolescents, and submaximal exercise at the same relative intensity also resulted in similar RQ values despite significantly greater average PO in the C group [CF = 38.7 (12.3-80) W and C = 67.8 (25.5-140) W, P = 0.039]. Excess postexercise oxygen consumption (EPOC) was greater in CF [2.79 (1.14-5.24) L O2] than C [1.46 (0.56-2.80) L O2] after submaximal exercise at a fixed PO (P = 0.036) but not after the relative exercise bout. CONCLUSIONS Habitual physical activity participation does not warrant adjustment of macronutrient intake ratios in adolescents with mild to moderate CF, but total caloric intake may need to be increased based on the level of EPOC and upon the intensity and the duration of the habitual activity.

Paternal high‐fat diet enhances offspring whole‐body insulin sensitivity and skeletal muscle insulin signaling early in life

Evidence suggests that paternal diet can predispose offspring to metabolic dysfunction. Despite this knowledge, little is known regarding the effects of paternal high‐fat feeding on offspring insulin sensitivity. The purpose of this study was to investigate for the first time the effects of paternal high‐fat feeding on whole‐body and skeletal muscle insulin action in young and adult offspring. At 4 weeks of age, founder C57BL6/N males (F0) were fed a high‐fat diet or control diet for 12 weeks and then bred with females on a control diet. Offspring (F1) were euthanized at 6 weeks, 6 months, or 12 months and insulin‐stimulated insulin signaling was measured ex vivo in isolated soleus muscle. At 6 weeks of age, paternal high fat offspring (HFO) had enhanced whole‐body insulin sensitivity (35%, P < 0.05), as well as, increased insulin‐stimulated skeletal muscle phosphorylation of Akt threonine 308 (70%, P < 0.05) and AS160 threonine 642 (80%, P < 0.05) compared to paternal control fed offspring (CFO), despite both offspring groups consuming standard chow. At 6 months of age, HFO had increased percent body fat compared to CFO (74%, P < 0.005) and whole‐body and skeletal muscle insulin signaling normalized to CFO. Body fat was inversely related with insulin signaling in HFO, but not CFO. These findings suggest that paternal high‐fat feeding contributes to enhanced whole‐body and skeletal muscle insulin sensitivity in HFO early in life; however, these benefits are lost by early adulthood, potentially due to premature increases in body fat.