Category: oxygen uptake


Abstract

Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α has been shown to play critical roles in regulating mitochondria biogenesis, respiration, and muscle oxidative phenotype. Furthermore, reductions in the expression of PGC-1α in muscle have been implicated in the pathogenesis of type 2 diabetes. To determine the effect of increased muscle-specific PGC-1α expression on muscle mitochondrial function and glucose and lipid metabolism in vivo, we examined body composition, energy balance, and liver and muscle insulin sensitivity by hyperinsulinemic-euglycemic clamp studies and muscle energetics by using 31P magnetic resonance spectroscopy in transgenic mice. Increased expression of PGC-1α in muscle resulted in a 2.4-fold increase in mitochondrial density, which was associated with an ≈60% increase in the unidirectional rate of ATP synthesis. Surprisingly, there was no effect of increased muscle PGC-1α expression on whole-body energy expenditure, and PGC-1α transgenic mice were more prone to fat-induced insulin resistance because of decreased insulin-stimulated muscle glucose uptake. The reduced insulin-stimulated muscle glucose uptake could most likely be attributed to a relative increase in fatty acid delivery/triglyceride reesterfication, as reflected by increased expression of CD36, acyl-CoA:diacylglycerol acyltransferase1, and mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase, that may have exceeded mitochondrial fatty acid oxidation, resulting in increased intracellular lipid accumulation and an increase in the membrane to cytosol diacylglycerol content. This, in turn, caused activation of PKCθ, decreased insulin signaling at the level of insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, and skeletal muscle insulin resistance.

FULL ARTICLE: http://www.pnas.org/content/105/50/19926.full.pdf+html

 

Abstract

BACKGROUND:

Maximal oxygen uptake (VO(2max)) predicts mortality and is associated with endurance performance. Trained subjects have a high VO(2max) due to a high cardiac output and high metabolic capacity of skeletal muscles. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, a fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training increases PGC-1α in skeletal muscle, PGC-1α-mediated changes may contribute to the improvement of exercise capacity and VO(2max). There are three isoforms of PGC-1α mRNA. PGC-1α-b protein, whose amino terminus is different from PGC-1α-a protein, is a predominant PGC-1α isoform in response to exercise. We investigated whether alterations of skeletal muscle metabolism by overexpression of PGC-1α-b in skeletal muscle, but not heart, would increase VO(2max) and exercise capacity.

METHODOLOGY/PRINCIPAL FINDINGS:

Transgenic mice showed overexpression of PGC-1α-b protein in skeletal muscle but not in heart. Overexpression of PGC-1α-b promoted mitochondrial biogenesis 4-fold, increased the expression of fatty acid transporters, enhanced angiogenesis in skeletal muscle 1.4 to 2.7-fold, and promoted exercise capacity (expressed by maximum speed) by 35% and peak oxygen uptake by 20%. Across a broad range of either the absolute exercise intensity, or the same relative exercise intensities, lipid oxidation was always higher in the transgenic mice than wild-type littermates, suggesting that lipid is the predominant fuel source for exercise in the transgenic mice. However, muscle glycogen usage during exercise was absent in the transgenic mice.

CONCLUSIONS/SIGNIFICANCE:

Increased mitochondrial biogenesis, capillaries, and fatty acid transporters in skeletal muscles may contribute to improved exercise capacity via an increase in fatty acid utilization. Increases in PGC-1α-b protein or function might be a useful strategy for sedentary subjects to perform exercise efficiently, which would lead to prevention of life-style related diseases and increased lifespan.

FULL ARTICLE CAN BE READ HERE:  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234261/pdf/pone.0028290.pdf

 

Abstract

BACKGROUND:

Maximal oxygen uptake (VO(2max)) predicts mortality and is associated with endurance performance. Trained subjects have a high VO(2max) due to a high cardiac output and high metabolic capacity of skeletal muscles. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, a fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training increases PGC-1α in skeletal muscle, PGC-1α-mediated changes may contribute to the improvement of exercise capacity and VO(2max). There are three isoforms of PGC-1α mRNA. PGC-1α-b protein, whose amino terminus is different from PGC-1α-a protein, is a predominant PGC-1α isoform in response to exercise. We investigated whether alterations of skeletal muscle metabolism by overexpression of PGC-1α-b in skeletal muscle, but not heart, would increase VO(2max) and exercise capacity.

METHODOLOGY/PRINCIPAL FINDINGS:

Transgenic mice showed overexpression of PGC-1α-b protein in skeletal muscle but not in heart. Overexpression of PGC-1α-b promoted mitochondrial biogenesis 4-fold, increased the expression of fatty acid transporters, enhanced angiogenesis in skeletal muscle 1.4 to 2.7-fold, and promoted exercise capacity (expressed by maximum speed) by 35% and peak oxygen uptake by 20%. Across a broad range of either the absolute exercise intensity, or the same relative exercise intensities, lipid oxidation was always higher in the transgenic mice than wild-type littermates, suggesting that lipid is the predominant fuel source for exercise in the transgenic mice. However, muscle glycogen usage during exercise was absent in the transgenic mice.

CONCLUSIONS/SIGNIFICANCE:

Increased mitochondrial biogenesis, capillaries, and fatty acid transporters in skeletal muscles may contribute to improved exercise capacity via an increase in fatty acid utilization. Increases in PGC-1α-b protein or function might be a useful strategy for sedentary subjects to perform exercise efficiently, which would lead to prevention of life-style related diseases and increased lifespan.

Astrand Laboratory of Work Physiology, The Swedish School of Sport and Health Sciences, Stockholm, Sweden. mikael.mattsson@ki.se

Abstract

In this paper we report a reversed drift in heart rate (HR) but increased oxygen uptake (VO(2)) during ultra-endurance exercise. Nine well-trained male athletes performed 24-h exercise in a controlled laboratory setting, with alternating blocks of kayaking, running and cycling. Each block included 110 min of exercise and 10 min of rest, with an average work intensity of approximately 55% of respective VO(2peak). Blood samples were taken and HR and VO(2) measured every 6th hour during steady-state cycling at fixed work rate. As assumed HR was increased at 6 h by 15 +/- 6 beats/min compared with initial level (0 h). Thereafter the drift did not progress continuously, but instead unexpectedly returned toward initial values, although the plasma levels of catecholamines increased continuously during exercise. VO(2) was increased by 0.22 +/- 0.15 L/min (10%) at 6 h and 0.37 +/- 0.18 L/min (17%) at 12 h compared with 0 h, and thereafter remained stable. This implies an increased oxygen pulse (VO(2)/HR) by approximately 10% at the last half of the 24-h exercise compared with 0 h. Consequently, sole use of HR would give inaccurate estimates of exercise intensity and energy expenditure during endurance exercise lasting more than 6 h, and different patterns of cardiovascular drift need to be taken into account.

Chronic oral ingestion of l-carnitine and carbohydrate increases muscle carnitine content and alters muscle fuel metabolism during exercise in humans

Non-technical summary

After 30 years of endeavour, this is the first study to show that muscle carnitine content can be increased in humans by dietary means and, perhaps more importantly, that carnitine plays a dual role in skeletal muscle fuel metabolism that is exercise intensity dependent. Specifically, we have shown that increasing muscle total carnitine content reduces muscle carbohydrate use during low intensity exercise, consistent with an increase in muscle lipid utilisation. However, during high intensity exercise muscle carnitine loading results in a better matching of glycolytic, pyruvate dehydrogenase complex and mitochondrial flux, thereby reducing muscle anaerobic energy generation. Collectively, these metabolic effects resulted in a reduced perception of effort and increased work output during a validated exercise performance test. These findings have significant implications for athletic performance and pathophysiological conditions where fat oxidation is impaired or anaerobic ATP production is increased during exercise.

Abstract

We have previously shown that insulin increases muscle total carnitine (TC) content during acute i.v. l-carnitine infusion. Here we determined the effects of chronic l-carnitine and carbohydrate (CHO; to elevate serum insulin) ingestion on muscle TC content and exercise metabolism and performance in humans. On three visits, each separated by 12 weeks, 14 healthy male volunteers (age 25.9 ± 2.1 years, BMI 23.0 ± 0.8 kg m−2) performed an exercise test comprising 30 min cycling at 50% Graphic, 30 min at 80% Graphic, then a 30 min work output performance trial. Muscle biopsies were obtained at rest and after exercise at 50% and 80% Graphic on each occasion. Following visit one, volunteers ingested either 80 g of CHO (Control) or 2 g of l-carnitine-l-tartrate and 80 g of CHO (Carnitine) twice daily for 24 weeks in a randomised, double blind manner. All significant effects reported occurred after 24 weeks. Muscle TC increased from basal by 21% in Carnitine (P < 0.05), and was unchanged in Control. At 50% Graphic, the Carnitine group utilised 55% less muscle glycogen compared to Control (P < 0.05) and 31% less pyruvate dehydrogenase complex (PDC) activation compared to before supplementation (P < 0.05). Conversely, at 80% Graphic, muscle PDC activation was 38% higher (P < 0.05), acetylcarnitine content showed a trend to be 16% greater (P < 0.10), muscle lactate content was 44% lower (P < 0.05) and the muscle PCr/ATP ratio was better maintained (P < 0.05) in Carnitine compared to Control. The Carnitine group increased work output 11% from baseline in the performance trial, while Control showed no change. This is the first demonstration that human muscle TC can be increased by dietary means and results in muscle glycogen sparing during low intensity exercise (consistent with an increase in lipid utilisation) and a better matching of glycolytic, PDC and mitochondrial flux during high intensity exercise, thereby reducing muscle anaerobic ATP production. Furthermore, these changes were associated with an improvement in exercise performance.

Abbreviations 

CHO
carbohydrate
CoASH
free co-enzyme A
CPT1
carnitine palmitoyl-transferase 1
PDC
pyruvate dehydrogenase complex
TC
total carnitine
Graphic
maximal oxygen uptake
Introduction

More than 95% of the body’s carnitine pool is confined to skeletal muscle, where it fulfils two major metabolic roles. Firstly, in mitochondrial fatty acid translocation carnitine is a substrate for carnitine palmitoyl-transferase 1 (CPT1) (Fritz & McEwen, 1959; Fritz & Yue, 1963). Secondly, during high intensity exercise, the formation of acetylcarnitine is essential for the maintenance of a viable pool of free co-enzyme A (CoASH), thereby enabling PDC and TCA flux to continue (Childress & Sacktor, 1966; Harris et al. 1987; Constantin-Teodosiu et al. 1991a). Not surprisingly therefore, oral carnitine feeding has been advocated as an ergogenic aid, the main premise being that increasing muscle carnitine content will increase muscle fat oxidation and delay muscle glycogen depletion. However, we are aware of no study that has unequivocally shown carnitine feeding can impact on muscle fuel metabolism or exercise performance, which is undoubtedly attributable to carnitine ingestion (or indeed i.v. carnitine infusion) per se failing to increase muscle carnitine content (Barnett et al. 1994; Vukovich et al. 1994; Wächter et al. 2002; Stephens et al. 2006a).

In a series of i.v. infusion studies, we demonstrated that elevating serum insulin concentration in the presence of hypercarnitinaemia (550–600 μmol l−1) acutely increased muscle total carnitine (TC) content by ∼15% in humans (Stephens et al. 2006a,b). Furthermore, this increase in carnitine retention occurred only when serum insulin concentration was elevated above 50 mU l−1, which we confirmed could be achieved by combined carbohydrate (CHO) and l-carnitine feeding (94 g and 3 g, respectively), albeit at a much lower rate of retention (equating to a projected ∼0.1% increase in the muscle TC pool per day; Stephens et al. 2007). Assuming this effect of combined CHO and carnitine feeding on carnitine retention is sustainable and cumulative, it was calculated that about 24 weeks of feeding would be needed to increase skeletal muscle TC content to the same extent as acute i.v. carnitine and insulin infusion.

During low intensity exercise, when PDC activation (PDCa) and flux are relatively low, the principal role of carnitine will most likely be mitochondrial fatty acid translocation. Although it has been suggested that free carnitine only limits fat oxidation at exercise intensities >70% of maximal oxygen consumption (Graphic; van Loon et al. 2001), we demonstrated that an acute 15% increase in muscle carnitine content reduced insulin-mediated muscle glycolytic flux and PDCa compared to control. Furthermore, this was accompanied by a subsequent increase in muscle glycogen and long-chain acyl-co-enzyme A accumulation (Stephens et al. 2006b), pointing to a carnitine-mediated increase in muscle fatty acid oxidation and CHO storage. Free carnitine availability may therefore be limiting to mitochondrial fatty acid translocation even at rest and during low intensity exercise, and an increase in skeletal muscle TC content would be expected to augment fatty acid oxidation and decrease PDCa and glycogen use during low intensity exercise.

During high intensity exercise, the primary functional role of carnitine shifts towards acetyl group buffering (i.e. forming acetylcarnitine) and maintaining a pool of free CoASH which is essential for mitochondrial flux to continue (including the PDC reaction). However, during exercise of this nature there is still an increase in the acetyl-co-enzyme A (acetyl-CoA)/CoASH ratio, possibly due to the significant depletion (to <6 mmol (kg dry muscle)−1) of the free carnitine pool caused by acetylcarnitine formation (Harris et al. 1987; van Loon et al. 2001). Therefore, it is plausible that an increase in skeletal muscle TC content would provide more effective buffering of acetyl-CoA production during high intensity exercise, offsetting the increase in the acetyl-CoA/CoASH ratio, and thereby increasing PDC flux and mitochondrial ATP production. This in turn would reduce the contribution from glycolysis and PCr hydrolysis to ATP production, particularly during the rest to exercise transition period when inertia in mitochondrial ATP production is known to reside at the level of PDC activation and flux (Timmons et al. 1997, 1998; Howlett et al. 1999; Roberts et al. 2002, 2005). In addition, increasing PDC flux during high intensity exercise would be expected to decrease muscle lactic acid production which could translate to a positive effect on exercise performance by reducing muscle acidosis (Sahlin, 1992). In support of this stance, previous work from our laboratory has shown that pharmacological activation of PDC at rest using the pyruvate dehydrogenase kinase inhibitor, dichloroacetate, markedly reduced muscle lactate production and PCr hydrolysis during a subsequent 20 min period of intense muscle contraction in isolated and perfused canine skeletal muscle, resulting in a substantial improvement in tension development (Timmons et al. 1997).

Based upon the above information, it is logical to conclude that increasing skeletal muscle TC content could alter muscle fuel metabolism in at least two different ways during exercise, with the dominating role being dictated by the exercise intensity employed. With this in mind, we first aimed to determine whether chronic l-carnitine and CHO feeding to healthy male volunteers could increase skeletal muscle TC content in a manner similar to that which we have observed acutely under i.v. l-carnitine infusion and hyperinsulinaemic clamp conditions. Secondly, we hypothesised that any increase in muscle TC content would result in a blunting of PDCa and flux during low intensity exercise, causing a corresponding decrease in muscle glycogen utilisation. Thirdly, during high intensity exercise, when the primary functional role of carnitine switches to acetyl group buffering, we proposed that increasing muscle carnitine content would increase muscle PDC flux (and mitochondrial ATP delivery), thereby reducing anaerobic ATP production and muscle lactic acidosis during exercise. Finally, we hypothesised that these positive metabolic effects of muscle carnitine loading would improve high intensity exercise performance.

Methods

Human volunteers

Fourteen healthy, non-smoking, non-vegetarian recreational athletes (Graphic 51.6 ± 2.5 ml (kg body mass)−1, training 3–5 times per week in triathlon, cycling, running or swimming), aged 25.9 ± 2.1 years and with a body mass index (BMI) of 23.0 ± 0.8 kg m–2 participated in this study. Moderately trained recreational athletes were recruited as they were accustomed to ingesting CHO supplements. The study was approved by the University of Nottingham Medical School Ethics Committee in accordance with the Declaration of Helsinki. Prior to the study, each subject completed a routine medical screening and a general health questionnaire to ensure their suitability to take part. All gave their informed written consent to participate in the study and were aware that they were free to withdraw from the experiment at any point.

Pre-testing

Fourteen days before the trial, each subject’s Graphic was measured using an electronically braked cycle ergometer (Lode NV Instrumenten, Groningen, the Netherlands) and a continuous and incremental, exhaustive exercise protocol. Oxygen consumption was measured using an online gas analyser (Vmax; SensorMedics, Anaheim, CA, USA) and Graphic was confirmed during a repeat test 3 days later. Graphic was accepted when a plateau in oxygen consumption was achieved despite a further increase in workload. Once Graphic had been obtained, workloads to be used in subsequent experimental visits were calculated that would elicit 50% and 80% of Graphic. Subjects were familiarised with the experimental exercise protocol (which also allowed confirmation that the workloads were at the appropriate intensity to elicit 50% and 80% Graphic) at least 1 week prior to the beginning of subsequent experimental visits. A repeat Graphic test was also performed at least 1 week after the completion of the study to confirm no significant change in aerobic capacity had occurred over the course of the study.

Experimental protocol

Volunteers reported to the laboratory at 08.30 h on three occasions over a 24 week period, each visit being separated by 12 weeks. Subjects arrived after an overnight fast having abstained from strenuous exercise and alcohol consumption for at least 48 h, and caffeine for at least 24 h. On each visit, subjects performed the following experimental protocol. On arrival at the laboratory, subjects were weighed and then rested in a semi-supine position whilst a resting blood sample was collected from an antecubital vein (venipuncture) for blood glucose, serum insulin and plasma TC concentration measurements. Volunteers then exercised for 30 min on the cycle ergometer at a workload corresponding to 50% Graphic followed immediately by 30 min of exercise at a predetermined workload of 80% Graphic. During both bouts of exercise, a rating of perceived exertion (Borg Scale) was obtained every 10 min. Finally, immediately following the completion of exercise at 80% Graphic, subjects performed a 30 min work output performance test. This ‘all-out’ performance test involved using the ergometer hyperbolic mode function, where work output is dependent upon volitional cycling cadence. This performance test has been shown to be a more reliable measurement of endurance exercise performance than cycling at a fixed exercise workload to volitional exhaustion (Jeukendrup et al. 1996), and has been used previously in our laboratory to measure performance (Stephens et al. 2008).

Supplementation protocol

After the first experimental visit, subjects were allocated in a randomised, double blind manner to two experimental treatment groups. One group (n = 7) was instructed to consume 700 ml of a solution containing 80 g of orange-flavoured CHO polymer (Vitargo; Swecarb AB, Stockholm, Sweden) on two occasions each day for 168 days (Control), whilst the remaining group consumed 80 g of orange-flavoured CHO polymer containing 2.0 g of l-carnitine tartrate (1.36 g of l-carnitine; Carnipure™, Lonza Group Ltd, Basel, Switzerland) in the same volume of solution and at the same frequency (Carnitine). Volunteers were instructed to ingest the first supplement at breakfast time and the second 4 h later. This feeding protocol was based upon a regimen we have previously shown to increase whole-body carnitine retention over a 14 day period (Stephens et al. 2007). Volunteers were informed of the caloric content of the drinks (∼600 calories per day) and advised to replace their customary CHO supplement with the prescribed supplement and/or amend their diet accordingly to try and avoid weight gain. Volunteers were requested to record any side effects associated with supplementation over the 24 week protocol. None were reported by either group.

Sample collection and analysis

On each experimental study day, venous blood samples were collected whilst subjects rested in a semi-supine position. Following collection, blood glucose concentrations were determined immediately using an autoanalyser (YSI 2300 STATplus, Yellow Springs Instruments, Yellow Springs, OH, USA). Two millilitres of each basal resting blood sample was collected into lithium heparin containers, and following centrifugation (22,000 RCF at +4°C for 2 min) the plasma was snap frozen in liquid nitrogen and stored at −80°C until used to determine plasma TC concentration using a radioenzymatic assay (Cederblad et al. 1982). Finally, a further 2 ml of each basal resting blood sample was allowed to clot and following centrifugation (1,400 RCF at +4°C for 10 min) the serum was stored frozen at −80°C until used to determine insulin concentration using a commercially available radioimmunoassay kit (Coat-a-Count Insulin, Diagnostics Products Corporation, Los Angeles, CA, USA).

On each experimental visit, muscle biopsy samples were obtained from the vastus lateralis muscle at rest and within 5 s of the end of exercise at 50% and 80% Graphic (whilst subjects were seated on the cycle ergometer) using the percutaneous needle biopsy technique (Bergström, 1975). Muscle samples were immediately snap frozen in liquid nitrogen after removal from the limb. One portion of each biopsy sample was freeze dried and stored at −80°C, whilst the remainder was stored ‘wet’ in liquid nitrogen. Freeze-dried muscle was dissected free of visible blood and connective tissue, powdered and used for the determination of muscle free carnitine, acetylcarnitine and long-chain acylcarnitine using the radioenzymatic method described previously by Cederblad (Cederblad et al. 1990). Muscle ATP, phosphocreatine (PCr), free creatine, lactate and glycogen were also determined on freeze-dried muscle using the spectrophotometric method of Harris (Harris et al. 1974). Muscle TC was calculated as the sum of the three carnitine moieties, and was normalised for the highest total creatine content from each individual’s three biopsies of that visit, a procedure routinely carried out to minimise variability from non-muscle constituents (Stephens et al. 2006a,b). Muscle total creatine content was calculated as the sum of free creatine and PCr. Approximately 10 mg of the ‘wet’ muscle was used to determine PDCa, expressed as the rate of acetyl-CoA formation (mmol min−1 (kg wet muscle)−1 at 37°C) using methodology described previously by Constantin-Teodosiu et al. (1991b). In addition, maximal citrate synthase activity was determined spectrophotometrically on whole muscle homogenates based on the methods of Opie & Newsholme (1967); Zammit & Newsholme (1976) and expressed as mmol min−1 (kg wet muscle)−1.

Statistical analysis

A two-way ANOVA for repeated measures (time and treatment effects) was performed to detect differences within and between treatment groups separately for the three conditions (rest, 50% Graphic and 80% Graphic). When a significant time or treatment effect was observed a one-way ANOVA or t test was performed, respectively, to locate individual differences. Statistical significance was declared at P < 0.05. All the values presented in text, tables and figures represent mean ± the standard error of the mean (s.e.m.).

Results

Subject characteristics

Subject characteristics are displayed in Table 1. Body mass was not different between groups before supplementation. However, there was a 2.4 kg increase in body mass from basal in the Control group after 12 weeks of supplementation (P < 0.01), which remained elevated after 24 weeks (P < 0.05). The Carnitine group showed no change in body mass over the course of the study. Fasting venous blood glucose and serum insulin concentrations at baseline were not different between groups and did not change throughout the study (Table 1). Fasting plasma TC concentration was also not different between groups before supplementation. However, plasma TC concentration in the Carnitine group was greater after 12 and 24 weeks of supplementation when compared to the Control group (P < 0.05; Table 1). Perceived exertion during exercise at 50% Graphic did not differ between groups on any visit. The same was also true during exercise at 80% Graphic before and after 12 weeks of supplementation. However, after 24 weeks of supplementation perceived exertion was lower in the Carnitine group when compared to baseline (14.0 vs. 15.0, respectively; P < 0.05) and Control at 24 weeks (14.0 vs. 16.2, respectively; P < 0.05).

View this table:

Table 1. Subject characteristics before (0) and 12 and 24 weeks after twice daily oral ingestion of either 80 g of carbohydrate (Control; n = 7) or 80 g of carbohydrate containing 2 g l-carnitine tartrate (Carnitine; n = 7)

Skeletal muscle total carnitine content

Resting muscle TC content over the course of the study is shown in Fig. 1. There was no difference between or within groups before or after 12 weeks of supplementation. However, after 24 weeks muscle TC content was 30% greater in the Carnitine group compared to Control (P < 0.05), which represented a 21% increase from baseline in the Carnitine group (P < 0.05).

Figure 1  Total skeletal muscle carnitine content (calculated as the mean of 3 biopsies taken from each individual during a given visit) before (0) and 12 and 24 weeks after twice daily oral ingestion of either 80 g of carbohydrate (Control; n = 7) or 80 g of carbohydrate containing 2 g l-carnitine tartrate (Carnitine; n = 7). All values are means ± s.e.m.). Significantly different from Control: *P < 0.05. Significantly different from before supplementation (0): †P < 0.05.

Skeletal muscle metabolites

Absolute muscle metabolite values at rest and during exercise are presented in Table 2. From similar resting values, muscle PCr, glycogen, lactate, acetylcarnitine and free carnitine content changed by a similar magnitude during exercise at 50 and 80% Graphic before and after 12 weeks of supplementation in Control and Carnitine groups. However, following 24 weeks of supplementation there was a trend (P = 0.09) for resting free carnitine content to be 30% greater in the Carnitine group compared to Control, and there was a significant difference between groups in the metabolic response to both low and high intensity exercise. Following exercise at 50% Graphic, muscle glycogen content was 35% greater in the Carnitine group compared to Control (P < 0.05), which equated to 55% less glycogen being utilised during exercise (P < 0.05; Fig. 2A), and free carnitine was 78% greater in the Carnitine group when compared to Control (P < 0.01). Following exercise at 80% Graphic, muscle glycogen content was 71% greater in the Carnitine group compared to Control; however, this was attributable to the reduction in glycogen utilisation during the preceding exercise at 50% Graphic (see above), and accordingly there was no difference between groups in glycogen utilisation during exercise at 80% Graphic (Fig. 2A). However, muscle lactate content was 44% lower in the Carnitine group compared to Control (P < 0.05) following exercise at 80% Graphic, which translated into a marked reduction in muscle lactate accumulation during exercise (P < 0.05, Fig. 2B) and was accompanied by a trend (P < 0.10) for muscle acetylcarnitine and free carnitine content to be greater in the Carnitine group when compared to Control (16% and 63%, respectively). In addition, after 24 weeks of supplementation, the muscle PCr/ATP ratio in the Carnitine group was significantly greater than Control (P < 0.05) and baseline (P < 0.05) following exercise at 80% Graphic.

Figure 2  Skeletal muscle glycogen utilisation (A), lactate accumulation (B) and pyruvate dehydrogenase complex activation status (C) during 30 min of exercise at 50 and 80% Graphic before (0) and 12 and 24 weeks after twice daily oral ingestion of either 80 g of carbohydrate (Control; n = 7) or 80 g of carbohydrate containing 2 g l-carnitine tartrate (Carnitine; n = 7). All values are means ± s.e.m. Significantly different from corresponding Control: *P < 0.05. Significantly different from before supplementation (0): †P < 0.05.

View this table:

Table 2. Skeletal muscle metabolites at rest and following 30 min of exercise at 50 and 80%Graphicbefore (0) and 12 and 24 weeks after twice daily oral ingestion of either 80 g of carbohydrate (Control; n = 7) or 80 g of carbohydrate containing 2 g l-carnitine tartrate (Carnitine; n = 7)

Muscle PDCa

Muscle pyruvate dehydrogenase activation status (PDCa) following exercise is shown in Fig. 2C. Resting muscle PDCa was not different between treatment groups at any time-point, being maintained at ∼0.4 mmol acetyl-CoA min−1 (kg wet muscle)−1. Similarly, muscle PDCa following exercise at 50% Graphic was not different between treatment groups at baseline and 12 weeks; however, at 24 weeks PDCa was 31% lower than baseline in the Carnitine group (P < 0.05). PDCa following exercise at 80% Graphic was not different between treatment groups at baseline or after 12 weeks, but was 38% greater in the Carnitine group at 24 weeks when compared with Control (P < 0.05).

Muscle citrate synthase activity

Muscle citrate synthase activity was not different between Control or Carnitine groups at baseline (5.6 ± 0.9 and 5.0 ± 0.4 mmol min−1 (kg wet muscle)−1, respectively) or after 24 weeks of supplementation (5.4 ± 0.1 and 5.9 ± 0.7 mmol min−1 (kg wet muscle)−1, respectively), and there were also no differences over time.

Exercise performance

Work output (kJ) achieved in the exercise performance test is presented in Fig. 3. Performance was not different between groups before or after 12 weeks of supplementation. However, after 24 weeks work output was 35% greater in the Carnitine group compared to Control (P < 0.05), which represented an 11% increase from baseline (P < 0.05).

Figure 3  Work output generated during a 30 min ‘all-out’ exercise performance test performed immediately following 30 min of exercise at 50 and 80% Graphic before (0) and 12 and 24 weeks after twice daily oral ingestion of either 80 g of carbohydrate (Control; n = 7) or 80 g of carbohydrate containing 2 g l-carnitine tartrate (Carnitine; n = 7). All values are means ± s.e.m. Significantly different from Control: *P < 0.05. Significantly different from before supplementation (0): †P < 0.05.

Discussion

Despite over 30 years of research demonstrating the fundamental role of carnitine in regulating muscle fuel use, attempts to increase skeletal muscle TC content in humans via l-carnitine feeding have been unsuccessful (Barnett et al. 1994; Vukovich et al. 1994; Wächter et al. 2002). The present study is the first to demonstrate that muscle TC content can be increased by 21% in healthy human volunteers when l-carnitine is ingested for 24 weeks in combination with a CHO solution. Moreover, this increase in TC content had a profound effect on muscle fuel utilisation during exercise which was exercise intensity dependent and consistent with the reported dual role of carnitine in muscle fuel metabolism. Namely, during low intensity exercise muscle glycogen utilisation was halved (consistent with an increase in muscle lipid utilisation), whereas during high intensity exercise muscle lactate accumulation was substantially reduced and the muscle PCr/ATP ratio was better maintained, which probably resulted from the carnitine-mediated increase in PDC activation and flux observed at this workload. Finally, increasing skeletal muscle TC content was associated with a 35% improvement in work output over Control, which we propose resulted directly from the observed changes in muscle fuel metabolism.

A major role of carnitine in skeletal muscle is as a substrate for the CPT1-mediated translocation of fatty acids into mitochondria for subsequent β-oxidation (Fritz & McEwen, 1959; Fritz & Yue, 1963). We have previously shown that a 15% increase in muscle TC content resulted in the attenuation of insulin-induced increases in glycolytic flux and PDCa in healthy, resting volunteers, as well as a subsequent overnight increase in muscle glycogen storage. This effect was attributed to a carnitine-mediated increase in acetyl-CoA delivery from fat oxidation, which inhibited PDCa and diverted glucose uptake from oxidation towards storage, and therefore suggests that carnitine availability is limiting to the CPT1 reaction under insulin-stimulated conditions, even at rest (Stephens et al. 2006b). We therefore hypothesised that during low intensity exercise in the present study, when glycolytic and PDC flux are well matched, an increase in muscle free carnitine availability would have a similar effect, i.e. it would augment muscle lipid oxidation thereby blunting PDCa and glycolytic flux. Consistent with this hypothesis, we observed that the increase in muscle TC content after 24 weeks of supplementation was linked to a 55% reduction in muscle glycogen utilisation during exercise at 50% Graphic compared to Control. Furthermore, this was accompanied by muscle free carnitine content being ∼80% greater and PDCa being 31% lower during exercise compared to before supplementation, suggesting that a carnitine-mediated increase in lipid-derived acetyl-CoA inhibited PDCa (Pettit et al. 1975) and thereby reduced muscle CHO flux, which is consistent with The Randle Cycle (Randle et al. 1963). Free carnitine availability has been suggested to limit muscle fat oxidation in vivo in humans during intense exercise when its concentration declines below 6 mmol (kg dry muscle)−1 (∼2 mmol (l intracellular water)−1) (van Loon et al. 2001). However, during exercise at 50% Graphic in the present study muscle free carnitine concentration was 11 mmol (kg dry muscle)−1 (∼3.5 mmol (l intracellular water)−1), which is above the value reported by van Loon et al. (2001) and well above the reported Km of CPT1 for free carnitine (0.5 mmol l−1) (McGarry et al. 1983). Therefore, either the reported Km of carnitine for CPT1, generated via in vitro experiments (McGarry et al. 1983) is not transferable to the in vivo situation or, alternatively, although the cellular carnitine pool is thought to be predominantly (90%) cytosolic (Zammit, 1999), the availability of free carnitine to CPT1 is markedly lower than suggested from determination of free carnitine in whole muscle homogenates. An explanation for this may be that the known catalytic site of CPT1 is located within the contact sites of the outer mitochondrial membrane and therefore not entirely available to the cytosolic carnitine pool. An increase in mitochondrial content over the duration of the study could also explain the apparent increase in fat oxidation and glycogen sparing observed in the Carnitine group during exercise at 50% Graphic after 24 weeks. However, if this was the case it would be expected that citrate synthase activity and/or Graphic would have also increased in this group, which was not observed. These observations, together with the finding that there was no evidence of glycogen sparing during exercise at 80% Graphic in the Carnitine group, makes it highly unlikely that an increase in mitochondrial content occurred over the 24 weeks of supplementation.

Another widely documented function of carnitine is as an acetyl group buffer during conditions of high glycolytic and PDC flux. During high intensity exercise, when acetyl group production by the PDC reaction is in excess of its utilisation by the TCA cycle, free carnitine buffers against acetyl-CoA accumulation by forming acetylcarnitine in a reaction catalysed by carnitine acetyl transferase (CAT), thereby ensuring a viable supply of free CoASH to sustain TCA cycle flux (Childress & Sacktor, 1966; Harris et al. 1987; Constantin-Teodosiu et al. 1991a). In the context of this, another major finding of the present study was the marked reduction in muscle lactate accumulation during exercise at 80% Graphic in the carnitine-loaded state after 24 weeks of supplementation when compared to Control, an effect probably mediated by the greater PDCa (38%) and flux (as evidenced by the 16% greater acetylcarnitine content) observed compared to Control. While it is clear that after 24 weeks muscle lactate did not accumulate in the Carnitine group to any lesser of an extent than seen at baseline, it is important to note that the absence of a change occurred in the face of increased glycogenolysis in both groups, which resulted in an increased lactate accumulation in the Control group only, and is explained by the carnitine-mediated increase in PDCa and flux in the Carnitine group. Furthermore, the magnitude of cellular energy disturbance (as indicated by PCr/ATP ratio) was significantly reduced during exercise at 80% Graphic at 24 weeks in the Carnitine group when compared to baseline and Control at 24 weeks. In keeping with this observation, we, and others, have previously reported that inertia in mitochondrial ATP production during the rest to exercise transition is at least partly limited by PDC activation and flux, resulting in increased anaerobic ATP generation (Timmons et al. 1997, 1998; Howlett et al. 1999; Roberts et al. 2002, 2005).

Pyruvate dehydrogenase activation status is principally regulated by a covalent mechanism of competing pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatases (PDP), which inactivate and activate the PDC, respectively. Pyruvate dehydrogenase kinase and PDP are themselves subject to metabolic regulation, and Ca2+ has been suggested as the principal metabolic activator of PDC during exercise by stimulating PDP (Constantin-Teodosiu et al. 2004). Considering that subjects in the present study exercised at the same work intensity (80% Graphic) during each visit, it can be assumed that the increase in cellular Ca2+ concentration was equal between visits and accordingly a similar stimulatory effect on the PDC was exerted regardless of skeletal muscle TC content. However, PDC activation is also regulated by end-product inhibition, primarily by an increase in the acetyl-CoA/CoASH ratio which stimulates PDK (Cooper et al. 1975; Pettit et al. 1975). Thus, as well as blunting acetyl-CoA accumulation during intense exercise and augmenting PDC flux, increased acetyl group buffering in the Carnitine group may also have modulated a reduction in the acetyl-CoA/CoASH ratio, thereby explaining the greater PDCa in the carnitine-supplemented group at 80% Graphic following 24 weeks of carnitine supplementation compared with Control.

Given that the performance test used in this study is reproducible (Jeukendrup et al. 1996; Stephens et al. 2008), and all the volunteers were recreational athletes familiar with intense exercise, a major finding of the present study has to be that the increase in muscle TC content after 24 weeks of supplementation resulted in a 35% increase in work output compared to Control (and an 11% increase from baseline). We, and others, have previously demonstrated that complete activation of PDC in the resting state, by pharmacological inhibition of PDK, markedly reduced anaerobic energy production in canine and human skeletal muscle during subsequent intense contraction (Timmons et al. 1997, 1998; Howlett et al. 1999; Roberts et al. 2002, 2005), and resulted in a substantial improvement in muscle contractile function (Timmons et al. 1997). It would appear therefore that the 38% greater PDCa and associated flux during exercise at 80% Graphic in the carnitine-loaded state in the present study, coupled with the reduction in muscle lactate accumulation and lower perceived exertion during exercise compared with Control, positively impacted upon work output during the subsequent performance trial. Indeed, it is likely that these metabolic effects observed at 80% Graphic in the carnitine-loaded state continued on into the performance trial given that subjects were attempting to perform as much work as possible in the 30 min of exercise. In keeping with our observations, Brass and colleagues have shown that carnitine loading of rodent soleus muscle reduced fatigue by 25% during electrically evoked contraction (Brass et al. 1993). Furthermore, Coyle (1995) concluded that the ability to maintain a high steady-state Graphic with low muscle lactate content is a prerequisite for enhanced endurance exercise performance in elite athletes. Indeed, muscle lactic acidosis has been suggested as a primary cause of fatigue during high-intensity exercise (Sahlin, 1992), hence the efforts to increase muscle buffering capacity by β-alanine feeding (Hill et al. 2007) or establish pre-exercise metabolic alkalosis by sodium bicarbonate ingestion (Wilkes et al. 1983; McKenzie et al. 1986; Bird et al. 1995) to improve high intensity exercise performance in humans. Finally, given that muscle glycogen content was 147 mmol (kg dry muscle) −1 following exercise at 80% Graphic in the Control group, it is possible that glycogen availability may have limited performance in this group during the 30 min work output trial, which would not have been the case in the Carnitine group where glycogen was 250 mmol (kg dry muscle)−1. It cannot be ruled out therefore that at least some of the positive effect of muscle carnitine loading on exercise performance was attributable to the glycogen sparing that occurred during exercise at 50% Graphic. Whether the beneficial effects of muscle carnitine loading on high intensity exercise and exercise performance led to the better maintenance of body mass in the face of 24 weeks of additional daily caloric intake when compared to control (i.e. via a regular increased energy expenditure during exercise training) is an interesting notion, but cannot be determined from the present study and remains to be explored.

In summary, this is the first study to demonstrate that muscle carnitine content can be increased in humans by dietary means and, perhaps more importantly, that carnitine plays a dual role in skeletal muscle fuel metabolism that is exercise intensity dependent. Specifically, we have shown that increasing muscle TC content spares muscle glycogen during low intensity exercise (consistent with an increase in muscle lipid utilisation), but during high intensity exercise results in a better matching of glycolytic, PDC and mitochondrial flux, thereby reducing muscle anaerobic ATP production. Furthermore, these metabolic changes resulted in positive effects on perception of effort and work output using a validated exercise performance test. Collectively these findings have significant implications for athletic performance and pathophysiological conditions where fat oxidation is impaired or anaerobic ATP production is accelerated during exercise (Noland et al. 2009).

Myo-Inositol trispyrophosphate: a novel allosteric effector of hemoglobin with high permeation selectivity across the red blood cell plasma membrane.

Source

Institut de Science et d’Ingénierie Supramoléculaires, Université de Strasbourg, 8 allée Gaspard Monge, 67000 Strasbourg, France.

Abstract

myo-Inositol trispyrophosphate (ITPP), a novel membrane-permeant allosteric effector of hemoglobin (Hb), enhances the regulated oxygen release capacity of red blood cells, thus counteracting the effects of hypoxia in diseases such as cancer and cardiovascular ailments. ITPP-induced shifting of the oxygen-hemoglobin equilibrium curve in red blood cells (RBCs) was inhibited by DIDS and NAP-taurine, indicating that band 3 protein, an anion transporter mainly localized on the RBC membrane, allows ITPP entry into RBCs. The maximum intracellular concentration of ITPP, determined by ion chromatography, was 5.5×10(-3) M, whereas a drop in concentration to the limit of detection was observed in NAP-taurine-treated RBCs. The dissociation constant of ITPP binding to RBC ghosts was found to be 1.72×10(-5) M. All data obtained indicate that ITPP uptake is mediated by band 3 protein and is thus highly tissue-selective towards RBCs, a feature of major importance for its potential therapeutic use.

The effect of pentoxifylline on the flow properties of human blood

Abstract

Recent investigations have revealed that erythrocytes from patients with chronic arterial occlusive disease are significantly less deformable than red blood cells from healthy subjects. The influence of pentoxifylline on red blood cell fluidity was measured by a standard filtration technique using 8 micron membrane filters. Impaired deformability of erythrocytes was significantly improved in patients suffering from peripheral vascular disorders following intravenous injection of 200 mg pentoxifylline. Studies on reduced red cell deformability induced by hyperosmolarity in vitro showed that pentoxifylline (4 and 20 microgram/ml) produced a dose-dependent improvement both in blood from healthy subjects and from patients with peripheral arterial occlusive disease. The results suggest that the positive therapeutic effect of pentoxifylline in peripheral arterial occlusive disease is mediated by improving red cell fluidity in the microcirculation.

PMID: 710175
[PubMed – indexed for MEDLINE]

Effect of pentoxiphylline on oxygen transport during hypothermia.

Source

Department of Anaesthesia, Harvard Medical School, Children’s Hospital, Boston, Massachusetts 02115.

Abstract

At least two investigators have demonstrated a reduction in O2 extraction during induced hypothermia (Cain and Bradley, J. Appl. Physiol. 55: 1713-1717, 1983; Schumacker et al., J. Appl. Physiol. 63: 1246-1252, 1987). We hypothesized that administration of pentoxiphylline (PTX), a theobromine that lowers blood viscosity and has vasodilator effects, would increase O2 extraction during hypothermia. To test this hypothesis, we studied O2 transport in anesthetized, paralyzed, mechanically ventilated beagles exposed to hypoxic hypoxia during either 1) normothermia (38 degrees C), 2) hypothermia (30 degrees C), or 3) hypothermia + PTX (30 degrees C and PTX, 20 mg.kg-1.h-1). Measurements included arterial and mixed venous PO2, hemoglobin concentration and saturation, cardiac output, systemic vascular resistance (SVR), blood viscosity, and O2 consumption (VO2). Critical levels of O2 delivery (DO2, the product of arterial O2 content and cardiac output) were determined by a system of linear regression. Hypothermia significantly decreased base line cardiac output (-35%), DO2 (-37%), and VO2 (-45%), while increasing SVR and blood viscosity. Addition of PTX increased cardiac output (35%) and VO2 (14%), and returned SVR and blood viscosity to normothermic levels. Hypothermia alone failed to significantly reduce the critical level of DO2, but addition of PTX did [normothermia, 11.4 +/- 4.2 (SD) ml.kg-1.min-1; hypothermia, 9.3 +/- 3.6; hypothermia + PTX, 6.6 +/- 1.3; P less than 0.05, analysis of variance]. The O2 extraction ratio (VO2/DO2) at the critical level of DO2 was decreased during hypothermia alone (normothermia, 0.60 +/- 0.13; hypothermia, 0.42 +/- 0.16; hypothermia + PTX, 0.62 +/- 0.19; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 2917962 [PubMed – indexed for MEDLINE]

Pentoxifylline improves haemoglobin and interleukin-6 levels in chronic kidney disease.

Source

Department of Nephrology, Fremantle Hospital, Alma Street, Perth, WA 6160, Australia. paolo.ferrari@health.wa.gov.au

Abstract

AIM:

To assess whether pentoxifylline improves anaemia of chronic kidney disease (CKD) via suppression of interleukin-6 (IL-6) and improved iron mobilization.

BACKGROUND:

CKD patients may have elevated IL-6 and tumour necrosis factor alpha levels. These cytokines can increase hepcidin production, which in turn reduces iron release from macrophages resulting in reduced availability of iron for erythropoiesis. In experimental models, pentoxifylline was shown to reduce IL-6 expression.

METHODS:

We studied 14 patients with stages 4-5 CKD (glomerular filtration rate <30mL/min per 1.73 m(2)) due to non-inflammatory renal diseases. None of the patients had received immunosuppressive or erythropoietin-stimulating agents or parenteral iron. Patients had weekly blood tests for iron studies and cytokines during a control run-in period of 3 weeks and during 4 weeks of pentoxifylline treatment.

RESULTS:

Ten patients (eGFR 23 + or – 6 mL/min) completed the study. At the end of the run-in period average haemoglobin was 111 + or – 5 g/L, ferritin 92 + or – 26 microg/L, transferrin saturation 15 + or – 3% and circulating IL-6 10.6 + or – 3.8 pg/mL. Tumour necrosis factor alpha values were below threshold for detection. Treatment with pentoxifylline reduced circulating IL-6 (6.6 + or – 1.6 pg/mL, P < 0.01), increased transferrin saturation (20 + or – 5%, P < 0.003) and decreased serum ferritin (81 + or – 25 microg/L, P = NS). Haemoglobin increased after the second week of pentoxifylline, reaching 123 + or – 6 g/L by week 4 (P < 0.001).

CONCLUSIONS:

Pentoxifylline reduces circulating IL-6 and improves haemoglobin in non-inflammatory moderate to severe CKD. These changes are associated with changes in circulating transferrin saturation and ferritin, suggesting improved iron release. It is hypothesized that pentoxifylline improves iron disposition possibly through modulation of hepcidin.

PMID:
20470305
[PubMed – indexed for MEDLINE]

Effects of pentoxifylline on oxidative stress and levels of EGF and NO in blood of diabetic type-2 patients; a randomized, double-blind placebo-controlled clinical trial.

Source

Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, PO Box 14155-6451, Tehran, Iran.

Abstract

BACKGROUND:

As oxidative stress contributes to both progression and pathologic complications of diabetes and effective therapeutic strategies to prevent or delay the damage remain limited, the aim of the present study was to assess the efficacy of pentoxifylline in reducing of oxidative stress. Since there is a relationship between nitric oxide (NO), epidermal growth factor (EGF) and oxidative stress, we measured the effect of this drug on these parameters in comparison to placebo.

METHODS:

Thirty-nine patients with type-2 diabetes mellitus were randomized in a double blind, placebo-controlled clinical trial to receive either pentoxifylline 400 mg four times a day or placebo for 14 days. Blood samples were obtained at baseline and at the end of the study. Samples were analyzed for thiobarbituric reactive substances (TBARS) as a marker of lipid peroxidation, ferric reducing ability (total antioxidant power, TAP), EGF and NO levels.

RESULTS:

Pentoxifylline in comparison to placebo was effective (P < 0.05) in reduction of lipid peroxidation in plasma of the patients without significant effects on TAP, levels of EGF and NO in plasma.

CONCLUSION:

Adding of pentoxifylline to drug regimen of diabetic type-2 patients can be helpful. Exact mechanism of action of pentoxifylline in reduction of blood lipid peroxidation remains to be elucidated.

PMID:
            15932791 begin_of_the_skype_highlighting            15932791      end_of_the_skype_highlighting      
[PubMed – indexed for MEDLINE]

Erythropoietin activates mitochondrial biogenesis and couples red cell mass to mitochondrial mass in the heart

Source

Duke University Medical Center , 0570 CR II Building White Zone, 200 Trent Dr, Durham NC 27710, USA.

Abstract

RATIONALE:

Erythropoietin (EPO) is often administered to cardiac patients with anemia, particularly from chronic kidney disease, and stimulation of erythropoiesis may stabilize left ventricular and renal function by recruiting protective effects beyond the correction of anemia.

OBJECTIVE:

We examined the hypothesis that EPO receptor (EpoR) ligand-binding, which activates endothelial NO synthase (eNOS), regulates the prosurvival program of mitochondrial biogenesis in the heart.

METHODS AND RESULTS:

We investigated the effects of EPO on mitochondrial biogenesis over 14 days in healthy mice. Mice expressing a mitochondrial green fluorescent protein reporter construct demonstrated sharp increases in myocardial mitochondrial density after 3 days of EPO administration that peaked at 7 days and surpassed hepatic or renal effects and anteceded significant increases in blood hemoglobin content. Quantitatively, in wild-type mice, complex II activity, state 3 respiration, and mtDNA copy number increased significantly; also, resting energy expenditure and natural running speed improved, with no evidence of an increase in left ventricular mass index. Mechanistically, EPO activated cardiac mitochondrial biogenesis by enhancement of nuclear respiratory factor-1, PGC-1alpha (peroxisome proliferator-activated receptor gamma coactivator 1alpha), and mitochondrial transcription factor-A gene expression in wild-type but not in eNOS(-/-) or protein kinase B (Akt1)(-/-) mice. EpoR was required, because EpoR silencing in cardiomyocytes blocked EPO-mediated nuclear translocation of nuclear respiratory factor-1.

CONCLUSIONS:

These findings support a new physiological and protective role for EPO, acting through its cell surface receptor and eNOS-Akt1 signal transduction, in matching cardiac mitochondrial mass to the convective O(2) transport capacity as erythrocyte mass expands.

PMID:
20395592
[PubMed – indexed for MEDLINE]
PMCID: PMC2895561
[Available on 2011/6/11]

Pentoxifylline Improves Hemoglobin Levels in Patients with Erythropoietin-resistant Anemia in Renal Failure

Angela Cooper*,†, Ashraf Mikhail*, Mark W. Lethbridge, D. Michael Kemeny and Iain C. Macdougall*
Departments of *Renal Medicine and Immunology, GKT School of Medicine, King’s College Hospital, London, United Kingdom.

Correspondence to Dr. Iain Macdougall, Department of Renal Medicine, King’s College Hospital, Bessemer Road, London, SE5 9PJ, United Kingdom. Phone: 44-207-346-6234; Fax: 44-207-346-6472; E-mail: iain.macdougall@kingsch.nhs.uk

Abstract

ABSTRACT. It was hypothesized that pentoxifylline might improve the response to recombinant human erythropoietin (rh-Epo) in anemic renal failure patients. Sixteen patients with ESRD and rh-Epo-resistant anemia, defined by a hemoglobin of <10.7 g/dl for 6 mo before treatment and a rh-Epo dose of ≥12,000 IU/wk, were recruited. They were treated with oral pentoxifylline 400 mg o.d. for 4 mo. Ex vivo T cell generation of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) from the patients was assessed before treatment and 6 to 8 wk after therapy. A total of 12 of 16 patients completed the study. Before therapy, the 12 patients’ mean hemoglobin concentration was 9.5 ± 0.9 g/dl. After 4 mo of pentoxifylline treatment, the mean hemoglobin concentration increased to 11.7 ± 1.0 g/dl (P = 0.0001). Baseline ex vivo T cell expression of TNF-α decreased from 58% ± 11% to 31% ± 23% (P = 0.0007) after therapy. Likewise, IFN-γ expression decreased from 31% ± 10% to 13% ± 10% (P = 0.0002). Pentoxifylline therapy may significantly improve the hemoglobin response in patients with previously rh-Epo-resistant anemia in renal failure. This may occur due to inhibition of proinflammatory cytokine production, which could interfere with the effectiveness of rh-Epo.

Introduction

The widespread use of recombinant erythropoietin (rh-Epo) has transformed the management of anemia in ESRD. Hemoglobin concentration improves in 90% to 95% of patients treated. Nevertheless, there is a small but important minority of patients who show an inadequate response to rh-Epo, and in a subset of these, no obvious cause (such as iron deficiency) can be found (1). Failure to respond to rh-Epo may be due to enhanced immune activation, which is known to occur in renal failure patients (2,3). Some proinflammatory cytokines (IFN-γ, TNF-α, and IL-1) suppress erythropoiesis in vitro (4,5). We have recently shown that T cells from renal failure patients responding poorly to rh-Epo generate more IFN-γ and TNF-α compared with both good responders to rh-Epo and healthy controls (6).

Pentoxifylline has been used for more than 20 yr in the treatment of peripheral vascular disease because of its potent hemorrheological properties (7). Subsequently, pentoxifylline was found to have antiinflammatory properties, mediated via inhibition of phosphodiesterase (8). In vitro, pentoxifylline inhibits monocyte production of TNF-α (9) and T cell production of IFN-γ (10,11). TNF-α is thought to play a central role in the pathogenesis of many diseases, prompting the experimental use of pentoxifylline in a number of clinical trials. Beneficial effects have been reported in idiopathic dilated cardiomyopathy (12), childhood type 1 diabetes (13), and systemic vasculitis (14–16). Modest clinical effects have also been observed in rheumatoid arthritis (17). To date, however, this drug has not been tested in patients with rh-Epo-resistant anemia, and the aim of the study presented here was to test the hypothesis that pentoxifylline inhibits proinflammatory cytokine production in vivo, giving rise to enhanced erythropoiesis.

Patients and Methods

Study Patients
We recruited 16 patients with ESRD who exhibited a poor response to rh-Epo. These patients all had hemoglobin concentrations of ≤10.7 g/dl for 6 mo before treatment, despite receiving high doses of rh-Epo ≥12,000 IU/wk. The percentage of patients in our unit with hemoglobin levels of ≤10 g/dl and rh-Epo doses of ≥12,000 IU/wk is 10.4%. Known factors that might inhibit the patients’ response to rh-Epo had previously been excluded from our recruited subjects, including iron deficiency, underdialysis, hyperparathyroidism, and acute infection. One patient was receiving monthly blood transfusions. Eleven of 16 patients were undergoing hemodialysis, 4 of 16 patients were managed by peritoneal dialysis, and 1 patient had a failing renal transplant. The study was approved by the Research Ethics Committee of King’s College Hospital, and informed consent was obtained from all subjects.

Study Protocol
Patients satisfying the above inclusion criteria were prescribed 400 mg pentoxifylline once daily after baseline determination of hemoglobin and T cell cytokines. The patients were given oral pentoxifylline for 12 wk, and T cell cytokine generation was determined immediately before the first dose. Repeat measurement of the T cell cytokines was performed after treatment with pentoxifylline for 6 to 8 wk. The dose of pentoxifylline selected for this study is slightly lower than that used in other clinical trials of this drug (400 mg three times a day) (12,18) and in clinical practice for peripheral vascular disease (400 mg two to three times a day). This is because the drug may accumulate in renal failure, and once-daily therapy may also improve compliance. The patients’ complete blood count is normally checked monthly as part of their standard clinical treatment. This allowed the retrospective assessment of hemoglobin over the previous 6 mo and the monthly monitoring of hemoglobin while the patients received pentoxifylline therapy.

Cell Preparation and Culture Conditions
Blood from peritoneal dialysis patients was collected in the morning and from hemodialysis patients immediately before a dialysis session. PBMC were isolated as described previously (6). PBMC were cultured in the presence of ionomycin (400 ng/ml; Sigma Chemical, Poole, Dorset, UK) plus PMA (10 ng/ml; Sigma). The protein transport inhibitor, brefeldin-A (5 µg/ml; Sigma) was included in all cultures.

T Cell Cytokine Measurement by Flow Cytometry
The method developed by Jung et al. (19) and Prussin and Metcalfe (20) was used for cytokine determination. After 18 h culture (37°C, 5% CO2), the PBMC were washed twice with PBS (Life Technologies, Paisley, UK) containing 0.1% BSA (Sigma) (200 g, 5 min, 4°C). The cells were initially stained with mouse anti-human PerCP-conjugated CD3 (Becton Dickinson, Cowley, Oxford, UK). Control cell samples were labeled with the appropriate FITC-, PE-, and PerCP-labeled isotype-matched antibodies. The cells were incubated with the conjugated antibodies for 30 min at 4°C in the dark, washed with PBS containing 0.1% BSA, and fixed with 4% formaldehyde solution for 20 min at room temperature in the dark. Cells were washed to remove the fixative and then permeabilized with 0.5% saponin (Sigma) in PBS containing 1% BSA for 20 min at room temperature in the dark. After permeabilization, cells were washed (200 g, 5 min, 4°C), then stained for intracellular cytokines with either PE-labeled TNF-α or FITC-labeled IFN-γ monoclonal antibodies (Becton Dickinson) (30 min, room temperature, in the dark). Cells were washed with 0.5% saponin in PBS containing 1% BSA and resuspended in 1% paraformaldehyde (Sigma), ready for flow cytometric analysis.

The labeled PBMC were analyzed on the same day as staining. Measurements were performed on a Becton Dickinson FACScan flow cytometer by CellQuest software 3.1. Initial gating was performed by using forward and side scatter to identify T lymphocytes. For each specimen, 20,000 events in the CD3 gate were collected. The percentage of CD3 cells positive for a cytokine was calculated by dividing the number of events in the CD3 positive/cytokine positive quadrant by the sum of events in both the CD3 positive/cytokine positive quadrant and the CD3 positive/cytokine negative quadrant.

Statistical Analyses
Statistical analyses were carried out by Prism version 3.0 statistical software (GraphPad Software, San Diego, CA; http://www.grphad.com). Results are expressed as mean ± SD. Differences between the pre- and posttreatment variables were analyzed by Student’s paired t test. Differences between demographic data of recruited patients and the renal unit patients were analyzed by the Mann-Whitney test. Correlation analysis was carried out by Spearman’s rank correlation. Results were considered significant at P < 0.05.

Results

Effect of Pentoxifylline on Hemoglobin Concentration
A total of 12 of 16 patients who started the study continued therapy for 4 mo. Two patients were noncompliant, one patient developed nausea, and one patient developed confusion unrelated to pentoxifylline therapy. Table 1 shows the demographic and laboratory data for the 12 patients who completed the study. The patients’ ferritin levels ranged from 184 to 1215 µg/L (median, 390 µg/L). It is unlikely that severe iron deficiency was present and responsible for the patients’ poor response to erythropoietin. The age of our patients was not dissimilar from the age of patients in our renal unit (54 ± 10 yr versus 60 ± 16 yr, P = 0.1069, respectively), whereas the rh-Epo dose was significantly different (study patients: 294 ± 125 IU/kg/wk versus renal unit patients: 104 ± 91 IU/kg/wk, P < 0.0001).

Table 1. Demographic and laboratory data of patients on pentoxifylline adjuvant therapya

Hemoglobin levels increased significantly from 9.5 ± 0.9 g/dl at the start of treatment to 11.7 ± 1.0 g/dl (P = 0.0001) after 4 mo of pentoxifylline therapy (Figure 1). These patients had refractory anemia for at least 6 mo before receiving pentoxifylline (Figure 1). One patient (patient 2, Table 1) was undergoing monthly blood transfusions before pentoxifylline therapy, but these became unnecessary when the patient’s hemoglobin increased to 11.7 g/dl.

Figure 1. Effect of pentoxifylline therapy on hemoglobin levels in 12 patients with a poor response to erythropoietic therapy, represented as a box and whisker plot. The horizontal line in the middle shows the median (50th percentile). The top and bottom of the box show the 75th and 25th percentiles, respectively. The whiskers show the maximum and minimum values. Hemoglobin values are presented monthly, 6 mo before pentoxifylline treatment and 4 mo while receiving pentoxifylline therapy

All but one of the patients were maintained on the same dose of subcutaneous rh-Epo during the study period. A dose reduction of rh-Epo was required for one patient (patient 11, Table 2), from 18,000 IU to 12,000 IU weekly, when his hemoglobin increased from 9.1 g/dl to 12.6 g/dl at 2 mo. For the 4 mo before receiving pentoxifylline, his hemoglobin (checked monthly) ranged from 8.7 to 9.2 g/dl, and after pentoxifylline, his hemoglobin was 12.6 g/dl at 2 mo, 12.9 g/dl at 3 mo, and 12.8 g/dl at 4 mo, despite the reduction in his dose of rh-Epo.

Table 2. White blood cell parameters of patients on pentoxifylline adjuvant therapya

Effect of Pentoxifylline on Circulating White Blood Cell Numbers
Table 2 shows the white blood cell parameters of the patients before and 4 mo after treatment. Pentoxifylline therapy had no effect on the numbers of circulating lymphocytes, neutrophils, monocytes, eosinophils, or basophils.

Effect of Pentoxifylline on Ex Vivo T Cell Cytokine Production
In the 12 patients studied, ex vivo T cell expression of TNF-α decreased from 58% ± 11% to 31% ± 23% (P = 0.0007) after 6 to 8 wk of pentoxifylline therapy. Figure 2 shows the individual pre- and postpentoxifylline TNF-α values. Similarly, IFN-γ expression decreased from 31% ± 10% to 13% ± 10% (P = 0.0002) (Figure 3). There was a significant correlation between change in hemoglobin (%) and TNF-α generation (%), as illustrated in Figure 4 (rs = 0.7145, P = 0.0118, n = 12). Although there was a trend in the change in hemoglobin (%) and IFN-γ generation (%), this did not reach statistical significance (Figure 5, rs = 0.4406, P = 0.1542, n = 12).

Figure 2. Effect of pentoxifylline therapy on T cell generation of TNF-α in 12 patients with a poor response to erythropoietic therapy. PBMC were isolated from whole blood, then stimulated in culture for 18 h before cytokine determination by flow cytometry. Each circle represents the percentage of T cells expressing TNF-α for each patient. The horizontal bar represents the mean values before and after therapy.

Figure 3. Effect of pentoxifylline therapy on T cell generation of IFN-γ in 12 patients with a poor response to erythropoietic therapy. PBMC were isolated from whole blood, then stimulated in culture for 18 h before cytokine determination by flow cytometry. Each circle represents the percentage of T cells expressing IFN-γ for each patient. The horizontal bars represent the means before and after therapy.

Figure 4. Effect of pentoxifylline therapy on percentage decrease of T cell generation of TNF-αversus percentage increase in hemoglobin levels in 12 patients with a poor response to erythropoietic therapy. Each square represents one patient.

Figure 5. Effect of pentoxifylline therapy on percentage decrease of T cell generation of IFN-γversus percentage increase in hemoglobin levels in 12 patients with a poor response to erythropoietic therapy. Each square represents one patient.

Discussion

This study describes a potential new use for an old drug. Pentoxifylline is licensed for the treatment of peripheral vascular disease, but its role as adjuvant therapy to rh-Epo has not previously been described. Nevertheless, like aspirin, it is a drug with ubiquitous properties, including anti-TNF-α (9) and anti-IFN-γ (10,11) actions, as well as antioxidant (21) and antiapoptotic effects (22). We selected pentoxifylline for investigation in rh-Epo-resistant patients because these patients show enhanced T cell generation of TNF-α and IFN-γ (6) compared with good responders to rh-Epo. Although this was conducted as an open-label study, the patients were carefully selected as those who had remained persistently anemic, with hemoglobin less than 10.7 g/dl despite receiving high doses of rh-Epo. This clinical state had persisted for a minimum of 6 mo in all patients, and indeed, most of them had failed to achieve their target hemoglobin level for a year before pentoxifylline therapy was initiated. It is therefore unlikely that the rise in hemoglobin in this cohort is the result of regression to the mean. One patient (patient 2) was heavily transfusion dependent before commencing pentoxifylline therapy and became transfusion-independent after treatment with this drug. Within 1 to 2 mo of initiating pentoxifylline, this cohort of patients showed a significant increase in hemoglobin concentration.

This study is to our knowledge the first demonstration that pentoxifylline therapy downregulates the ability of T cells to generate IFN-γ and TNF-α. Oral administration of pentoxifylline to healthy volunteers reduced TNF-α secretion from PBMC (18). In vitro studies on human whole blood have shown that pentoxifylline inhibits the production of IFN-γ (10,11). A study on purified human T cells reported that pentoxifylline reduced stimulated TNF-α, IL-5, and IL-10 production, but not IFN-γ secretion (23). In a murine model of allergic pulmonary inflammation, pentoxifylline treatment reduced IFN-γ levels in bronchial lavage and decreased expression of IFN-γ from stimulated spleen cells (24). This demonstrates that the drug can have an in vivo action on IFN-γ generation, which is consistent with our findings.

IFN-γ antagonizes the antiapoptotic effect of erythropoietin on erythroid colony-forming units (the developmental precursors of red blood cells in the bone marrow) (25). TNF-α inhibits erythropoiesis in vitro (5) via an indirect mechanism requiring IFN-{beta} (26). Both of these proinflammatory cytokines have been implicated in the anemia of chronic disease, a complication of inflammatory conditions and malignancy (27). In rheumatoid arthritis patients with anemia of chronic disease, administration of an anti-TNF-α antibody caused a significant rise in hemoglobin levels (28). There was a decrease in the numbers of apoptotic erythroid cells isolated from the bone marrow. Before therapy, anti-TNF-α antibody decreased the formation of erythroid colonies in vitro, but there was no effect after therapy. These findings support the concept that antagonizing the action of TNF-α is successful in treating inflammatory anemia. Anti-TNF-α therapy requires parenteral administration and has been associated with adverse skin reactions (29), increased risk of tuberculosis (30), and lymphoma (30). Pentoxifylline represents a more practical alternative for treatment of renal anemia because it is administered orally and has a low incidence of side effects. Pentoxifylline has a milder effect than other immunosuppressive drugs (such as cyclophosphamide) because the drug had no effect on the numbers of circulating lymphocytes. It is possible that the increase in proinflammatory cytokine generation in our rh-Epo-resistant patients is having a positive biologic effect; hence, inhibiting their generation may result in an adverse clinical action. In the context of renal anemia, we believe these cytokines have a negative action as they antagonize the proliferation of red blood cells (25,26).

Although this is an uncontrolled study, it was conducted under rigorous scientific conditions, and the temporal association with the commencement of pentoxifylline therapy suggests that this is a real effect. The patients in this study had experienced erythropoietin-resistant anemia for a minimum of 6 mo before receiving pentoxifylline, and some of them had shown inadequate hemoglobin levels for up to 1 yr before pentoxifylline therapy. No other interventions or clinical events coincided with the improvement in anemia, and there is a biologic rationale for this effect. The reduction in TNF-α generation from T cells significantly correlated with the rise in hemoglobin in the rh-Epo-resistant patients.

Although the reduction in both TNF-α and IFN-γ levels was associated with a rise in hemoglobin concentration, this study did not show that they were causally related. A poor response to erythropoietin is associated with increased mortality (31), and hemoglobin levels are inversely associated with death and hospitalization levels in patients with renal failure (32). Anemia is also thought to contribute to the risk of congestive heart failure in these patients (33). Correction of the hemoglobin levels with pentoxifylline may therefore have benefits on patient outcome. Because this project was an open-label interventional study, its findings should be interpreted cautiously, although the preliminary results warrant further investigation in a controlled, randomized study.

Leukocyte telomere length is preserved with aging in endurance exercise-trained adults and related to maximal aerobic capacity

Source

Department of Integrative Physiology, University of Colorado, Boulder, CO 80309, USA.

Abstract

Telomere length (TL), a measure of replicative senescence, decreases with aging, but the factors involved are incompletely understood. To determine if age-associated reductions in TL are related to habitual endurance exercise and maximal aerobic exercise capacity (maximal oxygen consumption, VO(2)max), we studied groups of young (18-32 years; n=15, 7 male) and older (55-72 years; n=15, 9 male) sedentary and young (n=10, 7 male) and older (n=17, 11 male) endurance exercise-trained healthy adults. Leukocyte TL (LTL) was shorter in the older (7059+/-141 bp) vs. young (8407+/-218) sedentary adults (P<0.01). LTL of the older endurance-trained adults (7992+/-169 bp) was approximately 900 bp greater than their sedentary peers (P<0.01) and was not significantly different (P=0.12) from young exercise-trained adults (8579+/-413). LTL was positively related to VO(2)max as a result of a significant association in older adults (r=0.44, P<0.01). Stepwise multiple regression analysis revealed that VO(2)max was the only independent predictor of LTL in the overall group. Our results indicate that LTL is preserved in healthy older adults who perform vigorous aerobic exercise and is positively related to maximal aerobic exercise capacity. This may represent a novel molecular mechanism underlying the “anti-aging” effects of maintaining high aerobic fitness.

2010 Elsevier Ireland Ltd. All rights reserved.

PMID:
20064545
[PubMed – indexed for MEDLINE]
PMCID: PMC2845985