Biomedical subjects

Michael Gaster

Publications and source records attributed to Michael Gaster.

16 recordsLinked to original sources

PubMed2006

Metabolic flexibility is conserved in diabetic myotubes.

The purpose of this study was to test the hypothesis that metabolic inflexibility is an intrinsic defect. Glucose and lipid oxidation were studied in human myotubes established from healthy lean and obese subjects and patients with type 2 diabetes (T2D). In lean myotubes, glucose oxidation is raised by increasing glucose concentrations (0-20 mmol/l) and acute insulin stimulation (P < 0.05), whereas it is inhibited by palmitate (PA). PA oxidation is raised by increasing PA concentrations (0-0.6 mmol/l), whereas 1.0 mmol/l PA inhibits its own oxidation (P < 0.05). Furthermore, PA oxidation is increased by acute insulin stimulation (P < 0.05) and inhibited by glucose. Even 0.05 mM PA and 2.5 mM glucose significantly reduce glucose and PA oxidation (P < 0.05), respectively. Glucose and PA oxidation are insulin-sensitive in myotubes established from lean (46% and 17% glucose and PA oxidation, respectively; P < 0.05 vs. basal), obese (31% and 14%; P < 0.05), and T2D (17% and 8%; P < 0.05) subjects. PA supplementation reduces both basal and insulin-stimulated glucose oxidation by 33-44% (P < 0.05), and myotubes are still insulin-sensitive in all three groups (P < 0.05). Therefore, the metabolic inflexibility described in obese and diabetic patients is not an intrinsic defect; rather, it is based on an extramuscular mechanism (i.e., the inability to vary extracellular fatty acid concentrations during insulin stimulation). Thus, skeletal muscles are metabolic-flexible per se.

Body Mass Index↗

PubMed2006

Muscle triacylglycerol and hormone-sensitive lipase activity in untrained and trained human muscles.

During exercise, triacylglycerol (TG) is recruited in skeletal muscles. We hypothesized that both muscle hormone-sensitive lipase (HSL) activity and TG recruitment would be higher in trained than in untrained subjects in response to prolonged exercise. Healthy male subjects (26 +/- 1 years, body moss index 23.3 +/- 0.5 kg m(-2)), either untrained (N = 8, VO(2max) 3.8 +/- 0.2 l min(-1)) or trained (N = 8, VO(2max) 5.1 +/- 0.1 l min(-1)), were studied. Before and after 3-h exercise (58 +/- 1% VO(2max)), a biopsy was taken. Muscle citrate synthase (32 +/- 2 vs. 47 +/- 6 mumol g(-1) min(-1) d.w.) and beta-hydroxy-acyl-CoA-dehydrogenase (38 +/- 3 vs. 52 +/- 5 mumol g(-1) min(-1) d.w.) activities were lower in untrained than in trained subjects (p < 0.05). Throughout the exercise, fat oxidation was higher in trained than in untrained subjects (p < 0.05). Muscle HSL activity was similar at rest (0.72 +/- 0.08 and 0.74 +/- 0.03 mU mg(-1) protein) and after exercise (0.71 +/- 0.1 and 0.68 +/- 0.03 mU mg(-1) protein) in untrained and trained subjects. At rest, the chemically determined muscle TG content (37 +/- 8 and 26 +/- 5 mmol g(-1) d.w.) was similar (p > 0.05), and after exercise it was unchanged in untrained and lower (p < 0.05) in trained subjects (41 +/- 9 and 10 +/- 2 mmol g((1) d.w.). Determined histochemically, TG was decreased (p < 0.05) after exercise in type I and II fibres. Depletion of TG was not different between fibre types in untrained, but tended to be higher (p = 0.07) in type I compared with type II fibres in trained muscles. In conclusion, HSL activity is similar in untrained and trained skeletal muscles both before and after prolonged exercise. However, the tendency to higher muscle TG recruitment during exercise in the trained subjects suggests a difference in the regulation of HSL or other lipases during exercise in trained compared with untrained subjects.

Adult↗

PubMed2006

Long-chain Acyl-CoA is not primarily increased in myotubes established from type 2 diabetic subjects.

Accumulation of intramuscular long-chain acyl-CoA esters (LCACoA) has previously in animal and human models been suggested to play an important role in lipid induced insulin resistance. The aim of this study was to examine whether myotubes established from type 2 diabetic (T2D) subjects and lean controls express differences in long-chain acyl-CoA esters (LCACoA) precultured under physiological conditions and during chronic exposure to palmitate (PA) and oleic acids (OA) with/without acute insulin stimulation. No significant differences were found between diabetic and control myotubes, neither in the total amount nor among individual LCA-CoA species during basal and acute insulin stimulation. LCA-CoA accumulated during exposure to palmitic acid but not during exposure to oleic acid. During PA and OA exposure, only palmitoyl-CoA, oleoyl-CoA and total LCA-CoA change. PA exposure increased the palmitoyl-CoA, whereas oleoyl-CoA was reduced and vice versa during OA exposure. No differences were found in the LCA-CoA level between T2D and control subjects, neither in the total amount nor in the individual specific LCA-CoA species during fatty acid exposure. Chronic (24 h), high PA, but not OA exposure induced insulin resistance at the level of glycogen synthesis in control subjects. These results indicate that (1) no primary defects are responsible for LCA-CoA accumulation in diabetic subjects; (2) LCA-CoA changes in vivo are partly adaptive to changes in the PA level and possibly other saturated fatty acids; and (3) PA induced insulin resistance may be mediated through an increased level of palmitoyl-CoA.

Acyl Coenzyme A↗

PubMed2006

Micro method for determination of nonesterified fatty acid in whole blood obtained by fingertip puncture.

Diagnostic tools for early identification of subjects at high risk for type 2 diabetes and other obesity-related disorders are important in prevention of these diseases. Nonesterified fatty acids (NEFAs) have been suggested to serve as a prediagnostic marker of diabetes and obesity-related disorders. In the current study, we developed a sensitive and reproducible micro method for quantification of NEFA in less than 10 microl whole blood. The method involves only two steps: (i) conversion of NEFA to fatty acid acyl-coenzyme A (acyl-CoA) esters using an acyl-CoA synthetase and (ii) quantification of the formed acyl-CoA esters with a fluorescent biosensor based on bovine acyl-CoA binding protein (ACBP). Lys50 of ACBP was mutagenized to a cysteine residue that was covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make a fluorescent acyl-CoA indicator (FACI-50). FACI-50 exhibits high fluorescence emission yield with maximum at 490 nm in the presence of CoA when excited at 387 nm. The addition of palmitoyl-CoA to a CoA-saturated FACI-50 lowered fluorescence emission by eightfold. Ethanol extract from 1 microl whole blood was incubated with ATP, CoA, and FACI-50. Following background fluorescence reading, NEFAs were converted to acyl-CoA by the acyl-CoA synthetase and the NEFA content was calculated from fluorescence emission changes using palmitic acid as external standard. The FACI-50 NEFA method was compared with two commercially available methods for quantification of NEFA.

Acyl Coenzyme A↗

PubMed2006

Triacylglycerol accumulation is not primarily affected in myotubes established from type 2 diabetic subjects.

In the present study, we investigated triacylglycerol (TAG) accumulation, glucose and fatty acid (FA) uptake, and glycogen synthesis (GS) in human myotubes from healthy, lean, and obese subjects with and without type 2 diabetes (T2D), exposed to increasing palmitate (PA) and oleate (OA) concentrations with/without high glucose and/or high insulin concentrations for 4 days. We showed that these myotubes expressed an increased TAG accumulation (P<0.001) without differences between groups. Chronically high insulin, but not high glucose concentrations, increases TAG accumulation by 25% (P<0.001). Inhibition of oxidative phosphorylation by antimycin A and oligomyin was followed by a reduced lipid oxidation (P<0.05) and increased TAG accumulation (P<0.05), but only in the presence of FAs. Both chronic PA and OA exposure reduced the insulin-mediated PA and OA uptake (fold change) (P<0.001), but could not induce insulin resistance at the level of glucose uptake, whereas high insulin concentrations induced insulin resistance (P<0.001). Chronic, high PA, but not OA, induced insulin resistance at the GS level in control subjects (P<0.05). The TAG content correlated negatively with insulin-stimulated FA uptake (P<0.001), but did not correlate with insulin-stimulated glucose uptake for PA or OA (P>0.05). These results indicate that (1) TAG accumulation is not primarily affected in skeletal muscle tissue of obese and T2D; (2) induced inhibition of oxidative phosphorylation is followed by TAG accumulation; (3) increasing FA and insulin availability, and reduced oxidative phosphorylation, and to a lesser extent glucose, are determinants for differences in intramyocellular TAG accumulation; (4) quantitative TAG content may not be the best marker for insulin resistance. Thus, increased TAG content in skeletal muscle of obese and T2D subjects is adaptive.

Cells, Cultured↗

PubMed2005

Reduced insulin-mediated citrate synthase activity in cultured skeletal muscle cells from patients with type 2 diabetes: evidence for an intrinsic oxidative enzyme defect.

In myotubes established from patients with type 2 diabetes (T2D), lipid oxidation and insulin-mediated glucose oxidation are reduced, whereas in myotubes from obese non-diabetic subjects, exposure to palmitate impairs insulin-mediated glucose oxidation. To determine the underlying mechanisms of these metabolic malfunctions, we studied mitochondrial respiration, uncoupled respiration and oxidative enzyme activities (citrate synthase (CS), 3-hydroxy-acyl-CoA-dehydrogenase activity (HAD)) before and after acute exposure to insulin and/or palmitate in myotubes established from healthy lean and obese subjects and T2D patients. Basal CS activity was lower (14%) in diabetic myotubes compared with myotubes from lean controls (P=0.03). Incubation with insulin (1 microM) for 4 h increased the CS activity (26-33%) in myotubes from both lean (P=0.02) and obese controls (P<0.001), but not from diabetic subjects. Co-incubation with palmitate (0.6 mM) for 4 h abolished the stimulatory effect of insulin on CS activity in non-diabetic myotubes. No differences were detected in mitochondrial respiration and HAD activity between myotubes from non-diabetic subjects and T2D patients, and none of these measures responded to high levels of insulin and/or palmitate. These results provide evidence for an intrinsic defect in CS activity, which may play a role in the pathogenesis of T2D. Moreover, the data suggest that insulin resistance at the CS level can be induced by exposure to high free fatty acid levels.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed2005

Differential utilization of saturated palmitate and unsaturated oleate: evidence from cultured myotubes.

We recently described a primarily reduced palmitate oxidation in myotubes established from type 2 diabetic subjects, whereas triacylglycerol (TAG) accumulation seemed to be adaptive. However, it is still uncertain whether these changes are similar for saturated and unsaturated fatty acids and whether high concentrations of glucose and/or insulin may change this picture. Studies of palmitic acid and oleic acid metabolism in human myotubes established from control and type 2 diabetic subjects under conditions of acute high concentrations of insulin and/or glucose may solve these questions. Total oleic acid and palmitic acid uptake in myotubes was increased during acute insulin stimulation (P < 0.01) but not under acute, high-glucose concentrations, and no differences were found between the groups. Type 2 diabetic myotubes expressed a reduced palmitic acid oxidation to carbon dioxide (P </= 0.04), whereas oleic acid oxidation showed no differences between myotubes from both groups. High glucose concentrations decreased oleic acid oxidation (P </= 0.03). Lipid distribution was not different in diabetic and control myotubes when palmitic acid and oleic acid incorporation into cellular lipids was compared. Myotubes that were exposed to palmitic acid showed an increased palmitic acid incorporation into diacylglycerol (DAG) and TAG compared with myotubes that were exposed to oleic acid (P < 0.05) expressing an increased intracellular free fatty acid (FFA) level (P < 0.05). Lipid distribution was not affected by high glucose, whereas insulin increased FFAs, DAG, and TAG (P < 0.05). De novo lipid synthesis from glucose in both diabetic and control myotubes was of the same magnitude independent of glucose and insulin concentrations. These results indicate that palmitic acid and oleic acid are utilized in the same pattern in diabetic and control myotubes even though palmitic acid oxidation is primarily reduced in diabetic cells. Palmitic acid and oleic acid are handled differently by myotubes: Palmitic acid seems to accumulate as DAG and TAG, whereas oleic acid accumulates as intracellular FFAs. These observations indicate that oleic acid is preferable as fatty acid as it accumulates to a lesser extent as DAG and TAG than palmitic acid. Neither acute hyperglycemia nor de novo lipid synthesis from glucose seems central to the TAG accumulation in obesity or type 2 diabetes.

Diabetes Mellitus, Type 2↗

PubMed2005

Skeletal muscle lipid accumulation in type 2 diabetes may involve the liver X receptor pathway.

Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism and are also involved in glucose metabolism. However, the functional role of LXRs in human skeletal muscle is at present unknown. This study demonstrates that chronic ligand activation of LXRs by a synthetic LXR agonist increases the uptake, distribution into complex cellular lipids, and oxidation of palmitate as well as the uptake and oxidation of glucose in cultured human skeletal muscle cells. Furthermore, the effect of the LXR agonist was additive to acute effects of insulin on palmitate uptake and metabolism. Consistently, activation of LXRs induced the expression of relevant genes: fatty acid translocase (CD36/FAT), glucose transporters (GLUT1 and -4), sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor-gamma, carnitine palmitoyltransferase-1, and uncoupling protein 2 and 3. Interestingly, in response to activation of LXRs, myotubes from patients with type 2 diabetes showed an elevated uptake and incorporation of palmitate into complex lipids but an absence of palmitate oxidation to CO(2). These results provide evidence for a functional role of LXRs in both lipid and glucose metabolism and energy uncoupling in human myotubes. Furthermore, these data suggest that increased intramyocellular lipid content in type 2 diabetic patients may involve an altered response to activation of components in the LXR pathway.

Anticholesteremic Agents↗

PubMed2004

Expression profiling of insulin action in human myotubes: induction of inflammatory and pro-angiogenic pathways in relationship with glycogen synthesis and type 2 diabetes.

Myotube cultures from patients with type 2 diabetes mellitus (T2DM) represent an experimental in vitro model of T2DM that offers a possibility to perform gene expression studies under standardized conditions. During a time-course of insulin stimulation (1 microM) at 5.5 mM glucose for 0 (no insulin), 0.5, 1, 2, 4, 8, and 24 h, mRNA contents were analyzed in human myotubes for each time point using Affymetrix DNA chip technology. Insulin treatment induced an inflammatory and pro-angiogenic response in the myotubes, with expression of early response factors followed by inflammatory chemokines, metabolic enzymes, and finally cell cycle regulating genes. One-hundred-forty-four genes were differentially expressed in myotubes from donors with type 2 diabetes compared with control subjects, including HSP70, apolipoprotein D/E, tropomyosin, myosin, and actin previously reported from in vivo studies of diabetic skeletal muscle. We conclude, (i) that insulin induces a time-dependent inflammatory and pro-angiogenic transcriptional response in cultured human myotubes, (ii) that myotubes in vitro retain a gene expression pattern specific for type 2 diabetes and sharing five genes with that of type 2 diabetic skeletal muscle in vivo, and (iii) that insulin, despite similar metabolic effects of glucose uptake and glycogen synthesis, regulates different pools of genes in skeletal muscle during in vivo and in vitro conditions.

Adult↗

PubMed2004

The reduced insulin-mediated glucose oxidation in skeletal muscle from type 2 diabetic subjects may be of genetic origin--evidence from cultured myotubes.

Several defects in response to insulin have been described in vivo and in vitro in type 2 diabetes: a decreased glucose transport, defective glucose oxidation and altered glycogen synthesis. At present, it is unknown whether glucose oxidation is primarily affected or secondarily affected by, e.g. increased free fatty acids (FFA). The aim of this study was to evaluate whether myotubes established from type 2 diabetic subjects express a primarily or a FFA-induced reduced insulin-mediated glucose oxidation. We have therefore investigated glucose oxidation under basal, physiological conditions and during acute insulin stimulation with/without FFA. We found that myotubes established from type 2 diabetic subjects express a reduced insulin-stimulated increase in glucose oxidation. Moreover, an acute exposure to FFA reduces insulin-mediated glucose oxidation without alterations in glucose uptake and glycogen synthesis. Thus, we conclude that the diminished increase in insulin-stimulated glucose oxidation seen in type 2 diabetic subjects in vivo may be of genetic origin. Moreover, the glucose-fatty acid cycle seems not to be crucial for the pathophysiology of insulin resistance.

Cells, Cultured↗

PubMed2004

The primary defect in glycogen synthase activity is not based on increased glycogen synthase kinase-3alpha activity in diabetic myotubes.

The mechanism responsible for the diminished activation of glycogen synthase (GS) in diabetic myotubes remains unclear, but may involve increased activity and/or expression of glycogen synthase kinase-3 (GSK-3). In myotubes established from type 2 diabetic and healthy control subjects we determined GS activity ratio, protein expression, and activity of GSK-3alpha and beta under basal and insulin-stimulated conditions when precultured in increasing insulin concentrations. In myotubes precultured at low insulin concentrations acute insulin stimulation increased GS activity more in control than in diabetic subjects, whereas the corresponding GSK-3alpha but not GSK-3beta activity was significantly reduced by acute insulin treatment in both groups. However, in myotubes precultured at high insulin concentrations the effect of insulin on GS and GSK-3alpha activity was blunted in both groups. The protein expression of GSK-3alpha or beta was unaffected. In conclusion, myotubes with a primary defect in GS activity express insulin responsive GSK-3alpha, suggesting that failure of insulin to decrease GS phosphorylation involves abnormal activity of another kinase or phosphatase.

Adult↗

PubMed2004

Correspondence analysis of microarray time-course data in case-control design.

Although different statistical approaches have been proposed for analyzing microarray time-course data, method for analyzing such data collected using the popular case-control design in clinical investigations has not been proposed perhaps due to the increased complexity for the existing parametric or non-parametric approaches. In this paper, we introduce a new multivariate data analyzing technique, the correspondence analysis, to analyze the high dimensional microarray time-course data in case-control design. We show, through an example on type 2 diabetes, how the nice features of the correspondence analysis can be use to explore the various time-course gene expression profiles that exist in the data. By coordinating and examining the projections on the reduced dimensions by both the genes and the time-course experiments, we are able to identify important genes and time-course patterns and make inferences on their biological relevance. Using the sample replicates, we propose a bootstrap procedure for inferring the significance of contributions on the leading dimensions by both the time-course experiments and the genes. Striking differences in the time-course patterns in the normal controls and diabetes patients have been revealed. In addition, the method also identifies genes that display similar or comparable time-course expression patterns shared by both the cases and the controls. We conclude that our correspondence analysis based approach can be a useful tool for analyzing high dimensional microarray data collected in clinical investigations.

Algorithms↗

PubMed2004

Strength training increases insulin-mediated glucose uptake, GLUT4 content, and insulin signaling in skeletal muscle in patients with type 2 diabetes.

Strength training represents an alternative to endurance training for patients with type 2 diabetes. Little is known about the effect on insulin action and key proteins in skeletal muscle, and the necessary volume of strength training is unknown. A total of 10 type 2 diabetic subjects and 7 healthy men (control subjects) strength-trained one leg three times per week for 6 weeks while the other leg remained untrained. Each session lasted no more than 30 min. After strength training, muscle biopsies were obtained, and an isoglycemic-hyperinsulinemic clamp combined with arterio-femoral venous catheterization of both legs was carried out. In general, qualitatively similar responses were obtained in both groups. During the clamp, leg blood flow was higher (P < 0.05) in trained versus untrained legs, but despite this, arterio-venous extraction glucose did not decrease in trained legs. Thus, leg glucose clearance was increased in trained legs (P < 0.05) and more than explained by increases in muscle mass. Strength training increased protein content of GLUT4, insulin receptor, protein kinase B-alpha/beta, glycogen synthase (GS), and GS total activity. In conclusion, we found that strength training for 30 min three times per week increases insulin action in skeletal muscle in both groups. The adaptation is attributable to local contraction-mediated mechanisms involving key proteins in the insulin signaling cascade.

Biological Transport↗

PubMed2004

Reduced lipid oxidation in skeletal muscle from type 2 diabetic subjects may be of genetic origin: evidence from cultured myotubes.

Insulin resistance in skeletal muscle in vivo is associated with reduced lipid oxidation and lipid accumulation. It is still uncertain whether changes in lipid metabolism represent an adaptive compensation at the cellular level or a direct expression of a genetic trait. Studies of palmitate metabolism in human myotubes established from control and type 2 diabetic subjects may solve this problem, as genetic defects are preserved and expressed in vitro. In this study, total uptake of palmitic acid was similar in myotubes established from both control and type 2 diabetic subjects under basal conditions and acute insulin stimulation. Myotubes established from diabetic subjects expressed a primary reduced palmitic acid oxidation to carbon dioxide with a concomitantly increased esterification of palmitic acid into phospholipids compared with control myotubes under basal conditions. Triacylglycerol (TAG) content and the incorporation of palmitic acid into diacylglycerol (DAG) and TAG at basal conditions did not vary between the groups. Acute insulin treatment significantly increased palmitate uptake and incorporation of palmitic acid into DAG and TAG in myotubes established from both study groups, but no difference was found in myotubes established from control and diabetic subjects. These results indicate that the reduced lipid oxidation in diabetic skeletal muscle in vivo may be of genetic origin; it also appears that TAG metabolism is not primarily affected in diabetic muscles under basal physiological conditions.

Blood Glucose↗

PubMed2003

Metabolic and genetic influence on glucose metabolism in type 2 diabetic subjects--experiences from relatives and twin studies.

Based on our investigations in first-degree relatives, in twins in general, and in monozygotic twins discordant for type 2 diabetes, we have studied the inheritance of glucose intolerance, insulin resistance and insulin secretion in order to evaluate the role of genes versus environment in the development of type 2 diabetes. Insulin resistance in type 2 diabetes is mainly linked to glucose disposal in skeletal muscle, i.e. reduced glycogen synthesis. In order to investigate the genetic component responsible for the reduced glycogen synthase activity and reduced glucose transport, we also investigated cultured myotubes based on in vivo skeletal muscle biopsies. The results obtained in our own studies are discussed in comparison with the international literature. We conclude that both genetic and environmental factors play a role in the development of type 2 diabetes (hyperglycaemia), and that only subjects who are genetically disposed to insulin resistance and who possess beta-cells which are unable to compensate for the degree of insulin resistance seem to develop type 2 diabetes. Variables of two gene alleles disposing to insulin resistance have been identified, and their role is discussed. The most important environmental factor seems to be obesity, but intrauterine malnutrition also plays a role. The cellular mechanism responsible for obesity/lipid-induced diabetes mellitus is discussed with specific emphasis on the role of accumulation of long-chain AcylCoA and triglycerides in liver, muscle and beta-cells.

Diabetes Mellitus, Type 2↗

PubMed2002

The diabetic phenotype is conserved in myotubes established from diabetic subjects: evidence for primary defects in glucose transport and glycogen synthase activity.

The most well-described defect in the pathophysiology of type 2 diabetes is reduced insulin-mediated glycogen synthesis in skeletal muscles. It is unclear whether this defect is primary or acquired secondary to dyslipidemia, hyperinsulinemia, or hyperglycemia. We determined the glycogen synthase (GS) activity; the content of glucose-6-phosphate, glucose, and glycogen; and the glucose transport in satellite cell cultures established from diabetic and control subjects. Myotubes were precultured in increasing insulin concentrations for 4 days and subsequently stimulated acutely by insulin. The present study shows that the basal glucose uptake as well as insulin-stimulated GS activity is reduced in satellite cell cultures established from patients with type 2 diabetes. Moreover, increasing insulin concentrations could compensate for the reduced GS activity to a certain extent, whereas chronic supraphysiological insulin concentrations induced insulin resistance in GS and glucose transport activity. Our data suggest that insulin resistance in patients with type 2 diabetes comprises at least two important defects under physiological insulin concentrations: a reduced glucose transport under basal conditions and a reduced GS activity under acute insulin stimulation, implicating a reduced glucose uptake in the fasting state and a diminished insulin-mediated storage of glucose as glycogen after a meal.

Adult↗