Altered plasma serine and 1-deoxydihydroceramide profiles are associated with diabetic neuropathy in type 2 diabetes and obesity.

Recent studies suggest that the accumulation of atypical, 1-deoxysphingolipids that lack the C1 hydroxyl group may be associated with diabetic neuropathy (DN). We hypothesized that specific plasma 1-deoxysphingolipids associate with DN severity, and that alterations in plasma serine and alanine associate with 1-deoxysphingolipid elevation in patients with type 2 diabetes (T2D). We examined individual 1-deoxysphingolipid species using LC/MS/MS in plasma samples from 75 individuals including lean controls (LC, n = 19), those with obesity (n = 19), obesity with T2D without DN (ob/T2D, n = 18), and obesity with T2D with DN (Ob/T2D/DN, n = 19). We observed a step wise increase in 1-deoxydihydroceramides across these four groups (spearman correlation coefficient r = 0.41, p = 0.0002). Mean total concentrations of 1-deoxydihydroceramides, and most individual 1-deoxydihydroceramide species, were higher in ob/T2D/DN versus LC group (8.939 vs. 5.195 pmol/100 µL for total 1-deoxydihydroceramides p = 0.005). No significant differences in 1-deoxydihydroceramides were observed between the ob/T2D and ob/T2D/DN groups. l-alanine was higher and l-serine lower in ob/T2D/DN versus LC groups (326.2 vs. 248.0 µM, p = 0.0086 and 70.2 vs. 89.8 µM, p = 0.0110), consistent with a potential contribution of these changes to the observed 1-deoxysphingolipids profiles. 1-deoxydihydroceramides correlated inversely with leg intraepidermal nerve fiber density (CC -0.40, p = 0.003). These findings indicate that 1-deoxydihydroceramides may be important biomarkers and/or mediators of DN.

Localisation and composition of skeletal muscle diacylglycerol predicts insulin resistance in humans.

AIMS/HYPOTHESIS: We sought to evaluate if the cellular localisation and molecular species of diacylglycerol (DAG) were related to insulin sensitivity in human skeletal muscle. METHODS: Healthy sedentary obese controls (Ob; n = 6; mean±SEM age 39.5 ± 2.3 years; mean ± SEM BMI 33.3 ± 1.4 kg/m(2)), individuals with type 2 diabetes (T2D; n = 6; age 44 ± 1.8 years; BMI 30.1 ± 2.3 kg/m(2)), and lean endurance-trained athletes (Ath; n = 10; age 35.4 ± 3.1 years; BMI 23.3 ± 0.8 kg/m(2)) were studied. Insulin sensitivity was determined using an IVGTT. Muscle biopsy specimens were taken after an overnight fast, fractionated using ultracentrifugation, and DAG species measured using liquid chromatography/MS/MS. RESULTS: Total muscle DAG concentration was higher in the Ob (mean ± SEM 13.3 ± 1.0 pmol/µg protein) and T2D (15.2 ± 1.0 pmol/µg protein) groups than the Ath group (10.0 ± 0.78 pmol/µg protein, p = 0.002). The majority (76-86%) DAG was localised in the membrane fraction for all groups, but was lowest in the Ath group (Ob, 86.2 ± 0.98%; T2D, 84.2 ± 1.2%; Ath, 75.9 ± 2.7%; p = 0.008). There were no differences in cytoplasmic DAG species (p > 0.12). Membrane DAG species C18:0/C20:4, Di-C16:0 and Di-C18:0 were significantly more abundant in the T2D group. Cytosolic DAG species were negatively related to activation of protein kinase C (PKC)ε but not PKCθ, whereas membrane DAG species were positively related to activation of PKCε, but not PKCθ. Only total membrane DAG (r = -0.624, p = 0.003) and Di-C18:0 (r = -0.595, p = 0.004) correlated with insulin sensitivity. Disaturated DAG species were significantly lower in the Ath group (p = 0.001), and significantly related to insulin sensitivity (r = -0.642, p = 0.002). CONCLUSIONS/INTERPRETATION: These data indicate that both cellular localisation and composition of DAG influence the relationship to insulin sensitivity. Our results suggest that only saturated DAG in skeletal muscle membranes are related to insulin resistance in humans.

Glycaemic variability is associated with coronary artery calcium in men with Type 1 diabetes: the Coronary Artery Calcification in Type 1 Diabetes study.

AIMS: We investigated coronary artery calcium in association with glucose levels and variability measured using continuous glucose monitoring in adults with Type 1 diabetes in the Coronary Artery Calcification in Type 1 Diabetes study. METHODS: Coronary artery calcium was measured by electron beam tomography. The presence of any coronary artery calcium was analysed with respect to glucose levels [mean(T) (mean glucose), % of values < 3.9 mmol/l, > 10 mmol/l and either < 3.9 or > 10 mmol/l] and glycaemic variability [sd(T) (sd of all glucose values); sd(dm) (sd of the daily mean glucose levels) and sd(hh:mm) (glucose sd for a specified time of day, over all days)] using 3-5 days of continuous glucose monitoring from 75 subjects (45 women, 30 men), age 42 ± 9 years (mean ± sd) and diabetes duration of 29 ± 8 years using logistic regression. RESULTS: We observed significant associations between coronary artery calcium and mean(T) (OR = 4.4, 95% CI 1.1-18.6), % of values > 10 mmol/l (OR = 5.5, 95% CI 1.3-22.6), % of measures < 3.9 or > 10 mmol/l (OR = 5.7, 95% CI 1.3-24.9), sd(T) (OR = 4.7, 95% CI 1.1-19.7), sd(dm) (OR = 6.0, 95% CI 1.2-30.4) and sd(hh:mm) (OR = 4.0, 95% CI 1.1-15.4), among men, but none of these variables were associated with the presence of coronary artery calcium in women. CONCLUSIONS: We report the novel finding that subclinical atherosclerosis is associated with glucose levels and variability in men with Type 1 diabetes. The relationship of coronary artery calcium and glucose variability in Type 1 diabetes, and potential gender differences in this association, deserve further study.

Knee extensor torque and perceived discomfort during symmetrical biphasic electromyostimulation.

The purpose of this study was to examine the effects of simultaneously delivering 2 channels of electromyostimulation (EMS) current using 2 different electrode arrangements. Ten men and 10 women university students had 4 reusable electrodes placed (2 proximal, 2 distal) medial and lateral on the quadriceps muscle group. Isokinetic voluntary peak torque (VPT) of the quadriceps was determined at 60 degrees x s(-1). A symmetrical biphasic square wave current was applied using 2 independent channels in either a parallel (P) or a crossed (X) electrode arrangement. Subjects increased the current until maximal tolerance was achieved. No significant differences in percent VPT or perceived discomfort (PD) were observed between men and women. Percent VPT was significantly greater using the X (57.2 +/- 11.3%) vs. the P (46.5 +/- 10.7%) pad placement; however, pad placement did not affect peak PD. Data from this study suggest that a 2-channel application of EMS using a crossed electrode arrangement provides greater knee extensor force without greater discomfort.

Endurance training, expression, and physiology of LDH, MCT1, and MCT4 in human skeletal muscle.

To evaluate the effects of endurance training on the expression of monocarboxylate transporters (MCT) in human vastus lateralis muscle, we compared the amounts of MCT1 and MCT4 in total muscle preparations (MU) and sarcolemma-enriched (SL) and mitochondria-enriched (MI) fractions before and after training. To determine if changes in muscle lactate release and oxidation were associated with training-induced changes in MCT expression, we correlated band densities in Western blots to lactate kinetics determined in vivo. Nine weeks of leg cycle endurance training [75% peak oxygen consumption (VO(2 peak))] increased muscle citrate synthase activity (+75%, P < 0.05) and percentage of type I myosin heavy chain (+50%, P < 0.05); percentage of MU lactate dehydrogenase-5 (M4) isozyme decreased (-12%, P < 0.05). MCT1 was detected in SL and MI fractions, and MCT4 was localized to the SL. Muscle MCT1 contents were consistent among subjects both before and after training; in contrast, MCT4 contents showed large interindividual variations. MCT1 amounts significantly increased in MU, SL, and MI after training (+90%, +60%, and +78%, respectively), whereas SL but not MU MCT4 content increased after training (+47%, P < 0.05). Mitochondrial MCT1 content was negatively correlated to net leg lactate release at rest (r = -0.85, P < 0.02). Sarcolemmal MCT1 and MCT4 contents correlated positively to net leg lactate release at 5 min of exercise at 65% VO(2 peak) (r = 0.76, P < 0.03 and r = 0. 86, P < 0.01, respectively). Results support the conclusions that 1) endurance training increases expression of MCT1 in muscle because of insertion of MCT1 into both sarcolemmal and mitochondrial membranes, 2) training has variable effects on sarcolemmal MCT4, and 3) both MCT1 and MCT4 participate in the cell-cell lactate shuttle, whereas MCT1 facilitates operation of the intracellular lactate shuttle.

Endurance training increases gluconeogenesis during rest and exercise in men.

The hypothesis that endurance training increases gluconeogenesis (GNG) during rest and exercise was evaluated. We determined glucose turnover with [6,6-(2)H]glucose and lactate incorporation into glucose by use of [3-(13)C]lactate during 1 h of cycle ergometry at two intensities [45 and 65% peak O(2) consumption (VO(2 peak))] before and after training [65% pretraining VO(2 peak)], same absolute workload (ABT), and 65% posttraining VO(2 peak), same relative intensity (RLT). Nine males (178.1 +/- 2.5 cm, 81.8 +/- 3.3 kg, 27.4 +/- 2.0 yr) trained for 9 wk on a cycle ergometer 5 times/wk for 1 h at 75% VO(2 peak). The power output that elicited 66.0 +/- 1.1% of VO(2 peak) pretraining elicited 54.0 +/- 1.7% posttraining. Rest and exercise arterial glucose concentrations were similar before and after training, regardless of exercise intensity. Arterial lactate concentration during exercise was significantly greater than at rest before and after training. Compared with 65% pretraining, arterial lactate concentration decreased at ABT (4.75 +/- 0.4 mM, 65% pretraining; 2.78 +/- 0.3 mM, ABT) and RLT (3.76 +/- 0.46 mM) (P < 0.05). At rest after training, the percentage of glucose rate of appearance (R(a)) from GNG more than doubled (1.98 +/- 0.5% pretraining; 5.45 +/- 1.3% posttraining), as did the rate of GNG (0.11 +/- 0.03 mg x kg(-1) x min(-1) pretraining, 0.24 +/- 0.06 mg x kg(-1) x min(-1) posttraining). During exercise after training, %glucose R(a) from GNG increased significantly at ABT (2.3 +/- 0.8% at 65% pre- vs. 7.6 +/- 2.1% posttraining) and RLT (6.1 +/- 1.5%), whereas GNG increased almost threefold (P < 0.05) at ABT (0.24 +/- 0.08 mg x kg(-1) x min(-1) 65% pre-, and 0.71 +/- 0.18 mg x kg(-1) x min(-1) posttraining) and RLT (0.75 +/- 0.26 mg x kg(-1) x min(-1)). We conclude that endurance training increases gluconeogenesis twofold at rest and threefold during exercise at given absolute and relative exercise intensities.

Active muscle and whole body lactate kinetics after endurance training in men.

We evaluated the hypotheses that endurance training decreases arterial lactate concentration ([lactate](a)) during continuous exercise by decreasing net lactate release () and appearance rates (R(a)) and increasing metabolic clearance rate (MCR). Measurements were made at two intensities before [45 and 65% peak O(2) consumption (VO(2 peak))] and after training [65% pretraining VO(2 peak), same absolute workload (ABT), and 65% posttraining VO(2 peak), same relative intensity (RLT)]. Nine men (27.4 +/- 2.0 yr) trained for 9 wk on a cycle ergometer, 5 times/wk at 75% VO(2 peak). Compared with the 65% VO(2 peak) pretraining condition (4.75 +/- 0.4 mM), [lactate](a) decreased at ABT (41%) and RLT (21%) (P < 0.05). decreased at ABT but not at RLT. Leg lactate uptake and oxidation were unchanged at ABT but increased at RLT. MCR was unchanged at ABT but increased at RLT. We conclude that 1) active skeletal muscle is not solely responsible for elevated [lactate](a); and 2) training increases leg lactate clearance, decreases whole body and leg lactate production at a given moderate-intensity power output, and increases both whole body and leg lactate clearance at a high relative power output.

Muscle net glucose uptake and glucose kinetics after endurance training in men.

We evaluated the hypotheses that alterations in glucose disposal rate (R(d)) due to endurance training are the result of changed net glucose uptake by active muscle and that blood glucose is shunted to working muscle during exercise requiring high relative power output. We studied leg net glucose uptake during 1 h of cycle ergometry at two intensities before training [45 and 65% of peak rate of oxygen consumption (VO(2 peak))] and after training [65% pretraining VO(2 peak), same absolute workload (ABT), and 65% posttraining VO(2 peak), same relative workload (RLT)]. Nine male subjects (178.1 +/- 2.5 cm, 81.8 +/- 3.3 kg, 27.4 +/- 2.0 yr) were tested before and after 9 wk of cycle ergometer training, five times a week at 75% VO(2 peak). The power output that elicited 66.0 +/- 1.1% of VO(2 peak) before training elicited 54.0 +/- 1.7% after training. Whole body glucose R(d) decreased posttraining at ABT (5.45 +/- 0.31 mg. kg(-1). min(-1) at 65% pretraining to 4.36 +/- 0.44 mg. kg(-1). min(-1)) but not at RLT (5.94 +/- 0.47 mg. kg(-1). min(-1)). Net glucose uptake was attenuated posttraining at ABT (1.87 +/- 0.42 mmol/min at 65% pretraining and 0.54 +/- 0.33 mmol/min) but not at RLT (2.25 +/- 0. 81 mmol/min). The decrease in leg net glucose uptake at ABT was of similar magnitude as the drop in glucose R(d) and thus could explain dampened glucose flux after training. Glycogen degradation also decreased posttraining at ABT but not RLT. Leg net glucose uptake accounted for 61% of blood glucose flux before training and 81% after training at the same relative (65% VO(2 peak)) workload and only 38% after training at ABT. We conclude that 1) alterations in active muscle glucose uptake with training determine changes in whole body glucose kinetics; 2) muscle glucose uptake decreases for a given, moderate intensity task after training; and 3) hard exercise (65% VO(2 peak)) promotes a glucose shunt from inactive tissues to active muscle.

Respiratory gas-exchange ratios during graded exercise in fed and fasted trained and untrained men.

We evaluated the hypotheses that endurance training increases relative lipid oxidation over a wide range of relative exercise intensities in fed and fasted states and that carbohydrate nutrition causes carbohydrate-derived fuels to predominate as energy sources during exercise. Pulmonary respiratory gas-exchange ratios [(RER) = CO2 production/O2 consumption (VO2)] were determined during four relative, graded exercise intensities in both fed and fasted states. Seven untrained (UT) men and seven category 2 and 3 US Cycling Federation cyclists (T) exercised in the morning in random order, with target power outputs of 20 and 40% peak VO2 (VO2 peak) for 2 h, 60% VO2 peak for 1.5 h, and 80% VO2 peak for a minimum of 30 min after either a 12-h overnight fast or 3 h after a standardized breakfast. Actual metabolic responses were 22 +/- 0.33, 40 +/- 0.31, 59 +/- 0.32, and 75 +/- 0.39% VO2 peak. T subjects showed significantly (P < 0.05) decreased RER compared with UT subjects at absolute workloads when fed and fasted. Fasting significantly decreased RER values compared with the fed state at 22, 40, and 59% VO2 peak in T and at 40 and 59% VO2 peak in UT subjects. Training decreased (P < 0.05) mean RER values compared with UT subjects at 22% VO2 peak when they fasted, and at 40% VO2 peak when fed or fasted, but not at higher relative exercise intensities in either nutritional state. Our results support the hypothesis that endurance training enhances lipid oxidation in men after a 12-h overnight fast at low relative exercise intensities (22 and 40% VO2 peak). However, a training effect on RER was not apparent at high relative exercise intensities (59 and 75% VO2 peak). Because most athletes train and compete at exercise intensities >40% maximal VO2, they will not oxidize a greater proportion of lipids compared with untrained subjects, regardless of nutritional state.

Evaluation of exercise and training on muscle lipid metabolism.

To evaluate the hypothesis that endurance training increases intramuscular triglyceride (IMTG) oxidation, we studied leg net free fatty acid (FFA) and glycerol exchange during 1 h of cycle ergometry at two intensities before training [45 and 65% of peak rate of oxygen consumption (V(O2) peak)] and after training [65% pretraining V(O2) peak, same absolute workload (ABT), and 65% posttraining V(O2) peak, same relative intensity (RLT)]. Nine male subjects (178.1 +/- 2.5 cm, 81.8 +/- 3.3 kg, 27.4 +/- 2.0 yr) were tested before and after 9 wk of cycle ergometer training, five times per week at 75% V(O2) peak. The power output that elicited 66.1 +/- 1.1% of V(O2) peak before training elicited 54.0 +/- 1.7% after training due to a 14.6 +/- 3.1% increase in V(O2) peak. Training significantly (P < 0.05) decreased pulmonary respiratory exchange ratio (RER) values at ABT (0.96 +/- 0.01 at 65% pre- vs. 0.93 +/- 0.01 posttraining) but not RLT (0.95 +/- 0.01). After training, leg respiratory quotient (RQ) was not significantly different at either ABT (0.98 +/- 0.02 pre- vs. 0.98 +/- 0.03 posttraining) or RLT (1.01 +/- 0.02). Net FFA uptake was increased at RLT but not ABT after training. FFA fractional extraction was not significantly different after training or at any exercise intensity. Net glycerol release, and therefore IMTG lipolysis calculated from three times net glycerol release, did not change from rest to exercise or at ABT but decreased at the same RLT after training. Muscle biopsies revealed minor muscle triglyceride changes during exercise. Simultaneous measurements of leg RQ, net FFA uptake, and glycerol release by working legs indicated no change in leg FFA oxidation, FFA uptake, or IMTG lipolysis during leg cycling exercise that elicits 65% pre- and 54% posttraining V(O2) peak. Training increases working muscle FFA uptake at 65% V(O2) peak, but high RER and RQ values at all work intensities indicate that FFA and IMTG are of secondary importance as fuels in moderate and greater-intensity exercise.

Regional differences in LV collagen accumulation and mature cross-linking after myocardial infarction in rats.

To determine the extent of and any regional differences in remodeling response of the extracellular matrix (ECM) to myocardial infarction (MI), moderate-to-large transmural infarcts were surgically produced in left ventricular (LV) free wall of rats. Animals were killed 13 wk after surgery. In comparison to age-matched controls, infarction was associated with an overall increase in heart weight, which included hypertrophy of both the right ventricle and LV. Although the remaining viable myocardium in LV free wall was significantly reduced, the interventricular septum was hypertrophied some 30% compared with control tissues (247 +/- 9 vs. 189 +/- 8 mg). Collagen concentration more than doubled in remaining viable free wall (8.92 +/- 0.59 vs. 3.95 +/- 0.25 mg/100 mg, P < 0.0001), and a smaller but still highly significant 27% increase occurred (P < 0.01) in the more remote septum. Degree of covalent cross-linking of collagen fibrils as assessed by hydroxylysylpyridinoline (HP) concentration also revealed regional differences in response of the ECM to infarction. Although HP concentration was increased 60% in viable free wall (P < 0.05) post-MI, it was unchanged in the septum. With respect to collagen characteristics of the transmural infarct per se, the scar exhibited still further increases in both collagen and HP concentrations compared with the already elevated values for these two parameters in viable free wall. The results indicate that any evaluation of the remodeling response of viable myocardium post-MI must include not only the myocyte but also the ECM, the principal component of which is collagen.