Søren Kristiansen

Palmitate transport and fatty acid transporters in red and white muscles

We performed studies 1) to investigate the kinetics of palmitate transport into giant sarcolemmal vesicles, 2) to determine whether the transport capacity is greater in red muscles than in white muscles, and 3) to determine whether putative long-chain fatty acid (LCFA) transporters are more abundant in red than in white muscles. For these studies we used giant sarcolemmal vesicles, which contained cytoplasmic fatty acid binding protein (FABPc), an intravesicular fatty acid sink. Intravesicular FABPcconcentrations were sufficiently high so as not to limit the uptake of palmitate under conditions of maximal palmitate uptake (i.e., 4.5-fold excess in white and 31.3-fold excess in red muscle vesicles). All of the palmitate taken up was recovered as unesterified palmitate. Palmitate uptake was reduced by phloretin (-50%), sulfo- N-succinimidyl oleate (-43%), anti-plasma membrane-bound FABP (FABPpm, -30%), trypsin (-45%), and when incubation temperature was lowered to 0°C (-70%). Palmitate uptake was also reduced by excess oleate (-65%), but not by excess octanoate or by glucose. Kinetic studies showed that maximal transport was 1.8-fold greater in red vesicles than in white vesicles. The Michaelis-Menten constant in both types of vesicles was ∼6 nM. Fatty acid transport protein mRNA and fatty acid translocase (FAT) mRNA were about fivefold greater in red muscles than in white muscles. FAT/CD36 and FABPpm proteins in red vesicles or in homogenates were greater than in white vesicles or homogenates ( P < 0.05). These studies provide the first evidence of a protein-mediated LCFA transport system in skeletal muscle. In this tissue, palmitate transport rates are greater in red than in white muscles because more LCFA transporters are available.We performed studies 1) to investigate the kinetics of palmitate transport into giant sarcolemmal vesicles, 2) to determine whether the transport capacity is greater in red muscles than in white muscles, and 3) to determine whether putative long-chain fatty acid (LCFA) transporters are more abundant in red than in white muscles. For these studies we used giant sarcolemmal vesicles, which contained cytoplasmic fatty acid binding protein (FABPc), an intravesicular fatty acid sink. Intravesicular FABPc concentrations were sufficiently high so as not to limit the uptake of palmitate under conditions of maximal palmitate uptake (i.e., 4.5-fold excess in white and 31.3-fold excess in red muscle vesicles). All of the palmitate taken up was recovered as unesterified palmitate. Palmitate uptake was reduced by phloretin (-50%), sulfo-N-succinimidyl oleate (-43%), anti-plasma membrane-bound FABP (FABPpm, -30%), trypsin (-45%), and when incubation temperature was lowered to 0 degrees C (-70%). Palmitate uptake was also reduced by excess oleate (-65%), but not by excess octanoate or by glucose. Kinetic studies showed that maximal transport was 1.8-fold greater in red vesicles than in white vesicles. The Michaelis-Menten constant in both types of vesicles was approximately 6 nM. Fatty acid transport protein mRNA and fatty acid translocase (FAT) mRNA were about fivefold greater in red muscles than in white muscles. FAT/CD36 and FABPpm proteins in red vesicles or in homogenates were greater than in white vesicles or homogenates (P < 0.05). These studies provide the first evidence of a protein-mediated LCFA transport system in skeletal muscle. In this tissue, palmitate transport rates are greater in red than in white muscles because more LCFA transporters are available.

Glycogen synthase localization and activity in rat skeletal muscle is strongly dependent on glycogen content.

1 The influence of muscle glycogen content on glycogen synthase (GS) localization and GS activity was investigated in skeletal muscle from male Wistar rats. 2 Two groups of rats were obtained, preconditioned with a combination of exercise and diet to obtain either high (HG) or low (LG) muscle glycogen content. The cellular distribution of GS was studied using subcellular fractionation and confocal microscopy of immunostained single muscle fibres. Stimulation of GS activity in HG and LG muscle was obtained with insulin or contractions in the perfused rat hindlimb model. 3 We demonstrate that GS translocates from a glycogen‐enriched membrane fraction to a cytoskeleton fraction when glycogen levels are decreased. Confocal microscopy supports the biochemical observations that the subcellular localization of GS is influenced by muscle glycogen content. GS was not found in the nucleus. 4 Investigation of the effect of glycogen content on GS activity in basal and insulin‐ and contraction‐stimulated muscle shows that glycogen has a strong inhibitory effect on GS activity. Our data demonstrate that glycogen is a more potent regulator of glycogen synthase activity than insulin. Furthermore we show that the contraction‐induced increase in GS activity is merely a result of a decrease in muscle glycogen content. 5 In conclusion, the present study shows that GS localization is influenced by muscle glycogen content and that not only basal but also insulin‐ and contraction‐stimulated GS activity is strongly regulated by glycogen content in skeletal muscle.

Eccentric exercise decreases maximal insulin action in humans: muscle and systemic effects.

1. Unaccustomed eccentric exercise decreases whole‐body insulin action in humans. To study the effects of one‐legged eccentric exercise on insulin action in muscle and systemically, the euglycaemic clamp technique combined with arterial and bilateral femoral venous catheterization was used. Seven subjects participated in two euglycaemic clamps, performed in random order. One clamp was preceded 2 days earlier by one‐legged eccentric exercise (post‐eccentric exercise clamp (PEC)) and one was without the prior exercise (control clamp (CC)). 2. During PEC the maximal insulin‐stimulated glucose uptake over the eccentric thigh was marginally lower when compared with the control thigh, (11.9%, 64.6 +/‐ 10.3 vs. 73.3 +/‐ 10.2 mumol kg‐1 min‐1, P = 0.08), whereas no inter‐thigh difference was observed at a submaximal insulin concentration. The glycogen concentration was lower in the eccentric thigh for all three clamp steps used (P < 0.05). The glucose transporter GLUT4 protein content was on average 39% lower (P < 0.05) in the eccentric thigh in the basal state, whereas the maximal activity of glycogen synthase was identical in the two thighs for all clamp steps. 3. The glucose infusion rate (GIR) necessary to maintain euglycaemia during maximal insulin stimulation was lower during PEC compared with CC (15.7%, 81.3 +/‐ 3.2 vs. 96.4 +/‐ 8.8 mumol kg‐1 min‐1, P < 0.05). 4. Our data show that 2 days after unaccustomed eccentric exercise, muscle and whole‐body insulin action is impaired at maximal but not submaximal concentrations. The local effect cannot account for the whole‐body effect, suggesting the release of a factor which decreases insulin responsiveness systemically.

Exercise metabolism in human skeletal muscle exposed to prior eccentric exercise

1 The effects of unaccustomed eccentric exercise on exercise metabolism during a subsequent bout of graded concentric exercise were investigated in seven healthy male subjects. Arterial and bilateral femoral venous catheters were inserted 2 days after eccentric exercise of one thigh (eccentric thigh) and blood samples were taken before and during graded two‐legged concentric knee‐extensor exercise. Muscle biopsies were obtained from the eccentric and control vastus lateralis before (rest) and after (post) the concentric exercise bout. 2 Maximal knee‐extensor concentric exercise capacity was decreased by an average of 23 % (P < 0.05) in the eccentric compared with the control thigh. 3 The resting muscle glycogen content was lower in the eccentric thigh than in the control thigh (402 ± 30 mmol (kg dry wt)−1vs. 515 ± 26 mmol (kg dry wt)−1, means ± s.e.m., P < 0.05), and following the two‐legged concentric exercise this difference substantially increased (190 ± 46 mmol (kg dry wt)−1vs. 379 ± 58 mmol (kg dry wt)−1, P < 0.05) despite identical power and duration of exercise with the two thighs. 4 There was no measurable difference in glucose uptake between the eccentric and control thigh before or during the graded two‐legged concentric exercise. Lactate release was higher from the eccentric thigh at rest and, just before termination of the exercise bout, release of lactate decreased from this thigh (suggesting decreased glycogenolysis), whereas no decrease was found from the contralateral control thigh. Lower glycerol release from the eccentric thigh during the first, lighter part of the exercise (P < 0.05) suggested impaired triacylglycerol breakdown. 5 At rest, sarcolemmal GLUT4 glucose transporter content and glucose transport were similar in the two thighs, and concentric exercise increased sarcolemmal GLUT4 content and glucose transport capacity similarly in the two thighs. 6 It is concluded that in muscle exposed to prior eccentric contractions, exercise at a given power output requires a higher relative workload than in undamaged muscle. This increases utilization of the decreased muscle glycogen stores, contributing to decreased endurance.

Sarcolemmal glucose transport and GLUT-4 translocation during exercise are diminished by endurance training

Glucose utilization during exercise of a given submaximal power output is decreased after endurance training. The aim of the present study was to elucidate the mechanisms behind this phenomenon by utilizing the sarcolemmal giant vesicle technique. Eight healthy young untrained men endurance trained one thigh for 3 wk. They then exercised both thighs simultaneously at the same work load (77% of peak O2 uptake of the untrained thigh) for 40 min. Training increased muscle GLUT-4 protein by 70% (P < 0.05). Glucose uptake during exercise was 38% lower (P < 0.05) in the trained (T) thigh than in the untrained (UT) thigh because of both a lower (P < 0.05) glucose extraction and blood flow in T. During exercise, sarcolemmal GLUT-4 protein content and glucose transport capacity increased significantly less in T than in UT muscle, and muscle glucose concentration was lower in T compared with UT (P < 0.05) at the end of exercise. It is concluded that, despite a large increase in muscle GLUT-4 with endurance training, exercise of a given submaximal power output increases muscle glucose uptake less in T than in UT muscle. It is suggested that the mechanism behind this phenomenon is blunted exercise-induced translocation of GLUT-4 to the sarcolemma, resulting in a blunted increase in sarcolemmal glucose transport in T muscle.Glucose utilization during exercise of a given submaximal power output is decreased after endurance training. The aim of the present study was to elucidate the mechanisms behind this phenomenon by utilizing the sarcolemmal giant vesicle technique. Eight healthy young untrained men endurance trained one thigh for 3 wk. They then exercised both thighs simultaneously at the same work load (77% of peak O2 uptake of the untrained thigh) for 40 min. Training increased muscle GLUT-4 protein by 70% ( P < 0.05). Glucose uptake during exercise was 38% lower ( P < 0.05) in the trained (T) thigh than in the untrained (UT) thigh because of both a lower ( P < 0.05) glucose extraction and blood flow in T. During exercise, sarcolemmal GLUT-4 protein content and glucose transport capacity increased significantly less in T than in UT muscle, and muscle glucose concentration was lower in T compared with UT ( P < 0.05) at the end of exercise. It is concluded that, despite a large increase in muscle GLUT-4 with endurance training, exercise of a given submaximal power output increases muscle glucose uptake less in T than in UT muscle. It is suggested that the mechanism behind this phenomenon is blunted exercise-induced translocation of GLUT-4 to the sarcolemma, resulting in a blunted increase in sarcolemmal glucose transport in T muscle.

MOLECULAR MECHANISMS INVOLVED IN GLUT4 TRANSLOCATION IN MUSCLE DURING INSULIN AND CONTRACTION STIMULATION

Studies in mammalian cells have established the existence of numerous intracellular signaling cascades that are critical intermediates in the regulation of various biological functions. Over the past few years considerable research has shown that many of these signaling proteins are expressed in skeletal muscle. However, the detailed mechanisms involved in the regulation of glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface membrane in response to insulin and contractions in skeletal muscle are not well understood. In the present essay we report three different approaches to unravel the GLUT4 translocation mechanism: 1. specific pertubation of the insulin and/or contraction signaling pathways; 2. characterization of the protein composition of GLUT4-containing vesicles with the expectation that knowledge of the constituent proteins of the vesicles may help in understanding their trafficking; 3. degree of co-immunolocalization of the GLUT4 glucose transporters with other membrane marker proteins assessed by immunofluorescense and electron microscopy.

GLUT5 expression and fructose transport in human skeletal muscle.

Biochemical and immunocytochemical studies have revealed that, in addition to GLUT1 and GLUT4, human skeletal muscle also expresses the GLUT5 hexose transporter. The subcellular distribution of GLUT5 is distinct from that of GLUT4, being localised exclusively in the sarcolemmal membrane. The substrate selectivity of GLUT5 is also considered to be different to that of GLUT1 and GLUT4 in that it operates primarily as a fructose transporter. Consistent with this suggestion studies in isolated human sarcolemmal vesicles have shown that fructose transport obeys saturable kinetics with a Vmax of 477 +/- 37 pmol.mg protein-1 min-1 and a Km of 8.3 +/- 1.2 mM. Unlike glucose uptake, fructose transport in sarcolemmal vesicles was not inhibited by cytochalasin B suggesting that glucose and fructose are unlikely to share a common route of entry into human muscle. Muscle exercise, which stimulates glucose uptake through the increased translocation of GLUT4 to the plasma membrane, does not increase fructose transport or sarcolemmal GLUT5 content. In contrast, muscle inactivity, induced as a result of limb immobilisation, caused a significant reduction in muscle GLUT4 expression with no detectable effects on GLUT5. The presence of a fructose transporter in human muscle is compatible with studies showing that this tissue can utilise fructose for both glycolysis and glycogenesis. However, the full extent to which provision of fructose via GLUT5 is important in meeting the energy requirements of human muscle during both physiological and pathophysiological circumstances remains an issue requiring further investigation.

Effect of vanadate on glucose transporter(GLUT4) intrinsic activity in skeletal muscle plasma membrane giant vesicles

Maximally effective concentrations of vanadate (a phosphotyrosine phosphatase inhibitor) increase glucose transport in muscle less than maximal insulin stimulation. This might be due to vanadate-induced decreased intrinsic activity of GLUT4 accompanying GLUT4 translocation. Thus, the effect of vanadate (NaVO3) on glucose transporter (GLUT4) intrinsic activity (V(max) = intrinsic activity x [GLUT4 protein]) was studied in muscle plasma membrane giant vesicles. Giant vesicles (average diameter 7.6 microns) were produced by collagenase treatment of rat skeletal muscle. The vesicles were incubated for 1.5 h with concentrations of vanadate ranging from 3 to 40 mmol l-1 at 34 degrees C before being used for determination of glucose transport. The dose-response curve showed that vanadate decreased the specific D-glucose uptake by a maximum of 70% compared with a control preparation. The vanadate-induced decrease in glucose uptake was not due to a decrease in number of vesicles. To further verify the apparent vanadate-induced decrease in GLUT4 intrinsic activity, the kinetics of glucose transport were also examined. In the presence of 10 mmol l-1 vanadate the V(max) and K(m) were decreased (P < 0.05, n = 6) 55% and 60%, respectively, compared with control. The plasma membrane GLUT4 protein content was not changed in response to vanadate. It is concluded that vanadate decreased glucose transport per GLUT4 (intrinsic activity). This finding suggests that regulation of glucose transport in skeletal muscle can involve changes in GLUT4 intrinsic activity.

Training effects on muscle glucose transport during exercise

Muscle glucose uptake is increased during exercise compared to rest. In general, muscle glucose uptake increases with increasing exercise intensity and duration. Whereas the arterio-venous concentration difference only increases 2-4-fold during exercise compared with rest the increase in muscle perfusion in 10-20 times and therefore quantitatively very important. During exercise the surface membrane glucose transport capacity increases in skeletal muscle primarily due to an increase in surface membrane GLUT4 protein content. Endurance training decreases muscle glucose uptake during exercise at a given absolute submaximal work-load despite a large increase in muscle GLUT4 protein content. We have shown that this decrease in glucose uptake at least in part is due to a blunted exercise-induced increase in sarcolemmal glucose transport capacity secondary to a blunted increase in sarcolemmal GLUT4 transporter number. Thus, endurance training leads to a marked reduction of the fraction of muscle GLUT4 that is translocated during a given submaximal exercise bout. Whether this is true also during exercise at higher intensities remains to be seen.

Methylated DNA for monitoring tumor growth and regression: How do we get there?

Abstract A wide range of protein cancer biomarkers is currently recommended in international guidelines for monitoring the growth and regression of solid tumors. However, a number of these markers are also present in low concentrations in blood obtained from healthy individuals and from patients with benign diseases. In contrast, evidence has accumulated that suggests that modified methylated DNA is strongly related to the cancer phenotype. The modifications found in modified methylated DNA include a global loss of methylation in the genomes of the tumor cells as well as focal hypermethylation of gene promoters. Because tumor cells naturally secrete DNA and upon cell death leak DNA, modified methylated DNA can be detected in blood, urine, sputum and other body fluids. At present international guidelines do not include recommendations for monitoring modified methylated DNA. The low level of evidence can partly be explained by incomplete collection of serial blood samples, by analytical challenges, and by lack of knowledge of how monitoring studies should be designed and how serial marker data obtained from individual patients should be interpreted. Here, we review the clinical validity and utility of methylated DNA for monitoring the activity of malignant disease.