Fuel metabolism during ultra-endurance exercise

 Cyclists either ingested 300 ml 100 g/l U-[¹⁴C] glucose solution every 30 min during 6 h rides at 55% of VO_2max (n=6) or they consumed unlabelled glucose and were infused with U-[¹⁴C] lactate (n=5). Maintenance of euglycaemia limited rises in circulating free fatty acids, noradrenaline and adrenaline concentrations to 0.9±0.1 mM, 27±4 nM and 2.0±0.5 nM, respectively, and sustained the oxidation of glucose and lactate. As muscle glycogen oxidation declined from 100±13 to 71±9 μmol/min/kg in the last 3 h of exercise, glucose and lactate oxidation and interconversion rates remained at approximately 60 and 50 and at about 4 and 5 μmol/min/kg, respectively. Continued high rates of carbohydrate oxidation led to a total oxidation of around 270 g glucose, 130 g plasma lactate and 530 g muscle glycogen. Oxidation of some 530 g of muscle glycogen far exceeded the predicted (about 250 g) initial glycogen content of the active muscles and suggested that there must have been a considerable diffusion of unlabelled lactate from glycogen breakdown in inactive muscle fibres to adjacent active muscle fibres via the interstitial fluid that did not equilibrate with ¹⁴C lactate in the circulation. Received: 19 September 1997 / Received after revision: 15 December 1997 / Accepted: 22 January 1998     

Combined effects of hypoxia and endurance training on lipid metabolism in rat skeletal muscle

Aim: To determine whether endurance training can counterbalance the negative effects of hypoxia on mitochondrial phosphorylation and expression of the long chain mitochondrial fatty acid transporter muscle carnitine palmitoyl transferase 1 (mCPT‐1). Methods: Male Wistar rats were exposed either to hypobaric hypoxia (at a simulated altitude of ≈4000 m, PIO₂ ≈ 90 mmHg) or to normoxia (sea level) for 5 weeks. In each environment, rats were randomly assigned to two groups. The trained group went through a 5‐week endurance training programme. The control group remained sedentary for the same time period. Muscle fatty acid oxidation capacity was evaluated after the 5‐week period on isolated mitochondria prepared from quadriceps muscles with the use of palmitoylcarnitine or pamitoylCoA + carnitine. Results: Chronic hypoxia decreased basal (V₀, ?31% with pamitoylCoA + carnitine and ?21% with palmitoylcarnitine, P < 0.05) and maximal (V_max, ?31% with pamitoylCoA + carnitine, P < 0.05) respiration rates, hydroxyacylCoA dehydrogenase activity (?48%, P < 0.05), mCPT‐1 activity index (?34%, P < 0.05) and mCPT‐1 protein content (?34%, P < 0.05). Five weeks of endurance training in hypoxia brought V₀, mCPT‐1 activity index and mCPT‐1 protein content values back to sedentary normoxic levels. Moreover, in the group trained in hypoxia, V_max reached a higher level than in the group that maintained a sedentary lifestyle in normoxia (24.2 nmol O₂· min^?1 · mg^?1 for hypoxic training vs. 19.9 nmol O₂ · min^?1 · mg^?1 for normoxic sedentarity, P < 0.05). Conclusion: Endurance training can attenuate chronic hypoxia‐induced impairments in mitochondrial fatty acid oxidation. This training effect seems mostly mediated by mCPT‐1 activity rather than by mCPT‐1 content.     

Long‐TE 1H MRS suggests that liver fat is more saturated than subcutaneous and visceral fat

Cross‐talk between adipose tissue and liver is disturbed in the metabolic syndrome. Moreover, the relative fatty acid composition of adipose and liver fat is poorly characterized. Long‐TE ¹H MRS can determine the unsaturation and polyunsaturation of adipose tissue. The aim of this study was to use long‐TE ¹H MRS to determine the composition of liver fat and its relation to adipose tissue composition. Sixteen subjects with increased liver fat (>5%) were recruited for the study. Using TE = 200 ms, we were able to resolve the olefinic (?CH, 5.3 ppm) and water (H₂O, 4.7 ppm) resonances in liver spectra and to obtain a repeatable estimate of liver fat unsaturation (coefficient of variation, 2.3%). With TE = 135 ms, the diallylic (?C? CH₂? C?, 2.8 ppm) resonance was detectable in subjects with a liver fat content above 15%. Long‐TE ¹H MRS was also used to determine the unsaturation in subcutaneous (n = 16) and visceral (n = 11) adipose tissue in the same subjects. Liver fat was more saturated (double bonds per fatty acid chain, 0.812 ± 0.022) than subcutaneous (double bonds per fatty acid chain, 0.862 ± 0.022, p < 0.0004) or visceral (double bonds per fatty acid chain, 0.865 ± 0.033, p < 0.0004) fat. Liver fat unsaturation correlated with subcutaneous unsaturation (R = 0.837, p < 0.0001) and visceral unsaturation (R = 0.879, p < 0.0004). The present study introduces a new noninvasive method for the assessment of the composition of liver fat. The results suggest that liver fat is more saturated than subcutaneous or visceral adipose tissue, which may be attributed to differences in de novo lipogenesis. Copyright © 2010 John Wiley & Sons, Ltd.     

Fatty acids are important for the Frank–Starling mechanism and Gregg effect but not for catecholamine response in isolated rat hearts

In some pathophysiological conditions myocardial metabolism can switch from mainly long chain fatty acid (LCFA) oxidation to mainly glucose oxidation. Whether the predominant fatty acid or glucose oxidation affects cardiac performance has not been defined. In a buffer perfused isovolumetrically contracting rat heart, oxidation of endogenous pool LCFA was avoided by inhibiting carnitine‐palmitoyl‐transferase I (CPT‐I) with oxfenicine (2 mm ). In order to restore fatty acid oxidation, hexanoate (1 mm ), which bypasses CPT‐I inhibition, was added to the perfusate. Three groups of hearts were subjected to either an increase in left ventricular volume (VV, +25%) or an increase in coronary flow (CF, +50%), or inotropic stimulation with isoproterenol (10^?8 and 10^?6 m ). The increase in VV (the Frank–Starling mechanism) increased rate–pressure product (RPP) by 21 ± 2% under control conditions, but only by 6 ± 2% during oxfenicine‐induced CPT‐I inhibition. The contractile response to changes in VV recovered after the addition of hexanoate. Similar results were obtained in hearts, in which an increase in CF was elicited (the Gregg phenomenon). Isoproterenol caused a similar increase in contractility regardless of the presence of oxfenicine or hexanoate. In all groups, a commensurate increase in oxygen consumption accompanied the increase in contractility. The fatty acid oxidation is necessary for an adequate contractile response of the isolated heart to increased pre‐load or flow, whereas the inotropic response to adrenergic β‐receptor stimulation is insensitive to changes in substrate availability.     

Characterization of hepatic fatty acids in mice with reduced liver fat by ultra‐short echo time 1H‐MRS at 14.1 T in vivo

Alterations in the hepatic lipid content (HLC) and fatty acid composition are associated with disruptions in whole body metabolism, both in humans and in rodent models, and can be non‐invasively assessed by ¹H‐MRS in vivo. We used ¹H‐MRS to characterize the hepatic fatty‐acyl chains of healthy mice and to follow changes caused by streptozotocin (STZ) injection. Using STEAM at 14.1 T with an ultra‐short T_E of 2.8 ms, confounding effects from T₂ relaxation and J‐coupling were avoided, allowing for accurate estimations of the contribution of unsaturated (UFA), saturated (SFA), mono‐unsaturated (MUFA) and poly‐unsaturated (PUFA) fatty‐acyl chains, number of double bonds, PU bonds and mean chain length. Compared with in vivo ¹H‐MRS, high resolution NMR performed in vitro in hepatic lipid extracts reported longer fatty‐acyl chains (18 versus 15 carbons) with a lower contribution from UFA (61 ± 1% versus 80 ± 5%) but a higher number of PU bonds per UFA (1.39 ± 0.03 versus 0.58 ± 0.08), driven by the presence of membrane species in the extracts. STZ injection caused a decrease of HLC (from 1.7 ± 0.3% to 0.7 ± 0.1%), an increase in the contribution of SFA (from 21 ± 2% to 45 ± 6%) and a reduction of the mean length (from 15 to 13 carbons) of cytosolic fatty‐acyl chains. In addition, SFAs were also likely to have increased in membrane lipids of STZ‐induced diabetic mice, along with a decrease of the mean chain length. These studies show the applicability of ¹H‐MRS in vivo to monitor changes in the composition of the hepatic fatty‐acyl chains in mice even when they exhibit reduced HLC, pointing to the value of this methodology to evaluate lipid‐lowering interventions in the scope of metabolic disorders. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantification of the triglyceride fatty acid composition with 3.0 T MRI

The aim of this work was to validate a sequential method for quantifying the triglyceride fatty acid composition with 3.0 T MRI. The image acquisition was performed with a 3D spoiled gradient multiple echo sequence. A specific phase correction algorithm was implemented to correct the native phase images for wrap, zero‐ and first‐order phase and rebuild the real part images. Then, using a model of a fat ¹H MR spectrum integrating nine components, the number of double bonds (ndb) and the number of methylene‐interrupted double bonds (nmidb) were derived. The chain length (CL) was obtained from these parameters using heuristic approximation. Validations were performed on different vegetable oils whose theoretical fatty acid composition was used as reference and in five human subjects. In vivo measurements were made in the liver and in the subcutaneous and visceral adipose tissues. Linear regressions showed strong correlations between ndb and nmidb quantified with MRI and the theoretical values calculated using oil composition. Mean ndb/nmidb/CL were 1.80 ± 0.25/0.51 ± 0.21/17.43 ± 0.07, 2.72 ± 0.31/0.94 ± 0.16/17.47 ± 0.08 and 2.53 ± 0.21/0.84 ± 0.14/17.43 ± 0.07 in the liver, subcutaneous and visceral adipose tissues respectively. The results suggest that the triglyceride fatty acid composition can be assessed in human fatty liver and adipose tissues with a clinically relevant MRI method at 3.0 T. Copyright © 2014 John Wiley & Sons, Ltd.     

Measuring mitochondrial metabolism in rat brain in vivo using MR Spectroscopy of hyperpolarized [2‐13C]pyruvate

Hyperpolarized [1‐¹³C]pyruvate ([1‐¹³C]Pyr) has been used to assess metabolism in healthy and diseased states, focusing on the downstream labeling of lactate (Lac), bicarbonate and alanine. Although hyperpolarized [2‐¹³C]Pyr, which retains the labeled carbon when Pyr is converted to acetyl‐coenzyme A, has been used successfully to assess mitochondrial metabolism in the heart, the application of [2‐¹³C]Pyr in the study of brain metabolism has been limited to date, with Lac being the only downstream metabolic product reported previously. In this study, single‐time‐point chemical shift imaging data were acquired from rat brain in vivo. [5‐¹³C]Glutamate, [1‐¹³C]acetylcarnitine and [1‐¹³C]citrate were detected in addition to resonances from [2‐¹³C]Pyr and [2‐¹³C]Lac. Brain metabolism was further investigated by infusing dichloroacetate, which upregulates Pyr flux to acetyl‐coenzyme A. After dichloroacetate administration, a 40% increase in [5‐¹³C]glutamate from 0.014 ± 0.004 to 0.020 ± 0.006 (p = 0.02), primarily from brain, and a trend to higher citrate (0.002 ± 0.001 to 0.004 ± 0.002) were detected, whereas [1‐¹³C]acetylcarnitine was increased in peripheral tissues. This study demonstrates, for the first time, that hyperpolarized [2‐¹³C]Pyr can be used for the in vivo investigation of mitochondrial function and tricarboxylic acid cycle metabolism in brain. Copyright © 2013 John Wiley & Sons, Ltd.     

Peroxisomal fatty acid oxidation capacity is more resistant to ischaemic and reperfusion injury than mitochondrial fatty acid oxidation capacity in feline hearts

We investigated ischaemic and postischaemic mitochondrial and peroxisomal fatty acid oxidation capacity, ATP levels and regional function in 40 anaesthetized open chest cats subjected to 10 or 40 min of regional myocardial ischaemia with or without 3 h of reperfusion (n=10 in each situation). Following 10 min of ischaemia, the mitochondrial fatty acid oxidation capacity measured in tissue extracts from ischaemic tissue (nmol min^-1 mg protein^-1) was reduced in both subepi- and subendocardium, but was normalized in reperfused tissue extracts from both wall layers (0.29±0.03 and 0.30±0.04 vs. 0.57±0.05 and 0.59±0.05, P<0.05). Peroxisomal fatty acid oxidation capacity in tissue extracts was unaffected by ischaemia and reperfusion. ATP levels and regional function measured in the LAD region was partly restored transmurally. After 40 min of LAD occlusion, mitochondrial fatty acid oxidation capacity was reduced, with higher activity in subepi- than in subendocardium (0.27±0.05 vs. 0.19±0.04, P<0.05). Reperfusion did not restore mitochondrial fatty acid oxidation capacity. Peroxisomal fatty acid oxidation capacity was increased in the ischaemic subendocardium compared with levels in non-ischaemic subendocardium (0.53±0.02 vs. 0.45±0.03, P<0.05), with normalization at the end of reperfusion. ATP levels were non-uniformly reduced during ischaemia and not repleted during reperfusion. Regional function recovered in circumferential segments but not in longitudinal segments following 40 min of ischaemia. In conclusion fatty acid oxidation enzymes seem to be more resistant to ischaemia in peroxisomes than in mitochondria. Mitochondrial fatty acid oxidation is fully reversible following shortlasting ischaemia, but remains depressed following prolonged ischaemia and reperfusion.     

T2 relaxation times of 13C metabolites in a rat hepatocellular carcinoma model measured in vivo using 13C‐MRS of hyperpolarized [1‐13C]pyruvate

A single‐voxel Carr‐Purcell‐Meibloom‐Gill sequence was developed to measure localized T₂ relaxation times of ¹³C‐labeled metabolites in vivo for the first time. Following hyperpolarized [1‐¹³C]pyruvate injections, pyruvate and its metabolic products, alanine and lactate, were observed in the liver of five rats with hepatocellular carcinoma and five healthy control rats. The T₂ relaxation times of alanine and lactate were both significantly longer in HCC tumors than in normal livers (p < 0.002). The HCC tumors also showed significantly higher alanine signal relative to the total ¹³C signal than normal livers (p < 0.006). The intra‐ and inter‐subject variations of the alanine T₂ relaxation time were 11% and 13%, respectively. The intra‐ and inter‐subject variations of the lactate T₂ relaxation time were 6% and 7%, respectively. The intra‐subject variability of alanine to total carbon ratio was 16% and the inter‐subject variability 28%. The intra‐subject variability of lactate to total carbon ratio was 14% and the inter‐subject variability 20%. The study results show that the signal level and relaxivity of [1‐¹³C]alanine may be promising biomarkers for HCC tumors. Its diagnostic values in HCC staging and treatment monitoring are yet to be explored. Copyright © 2010 John Wiley & Sons, Ltd.     

Ketone bodies effectively compete with glucose for neuronal acetyl‐CoA generation in rat hippocampal slices

Ketone bodies can be used for cerebral energy generation in situ, when their availability is increased as during fasting or ingestion of a ketogenic diet. However, it is not known how effectively ketone bodies compete with glucose, lactate, and pyruvate for energy generation in the brain parenchyma. Hence, the contributions of exogenous 5.0 mM [1‐¹³C]glucose and 1.0 mM [2‐¹³C]lactate + 0.1 mM pyruvate (combined [2‐¹³C]lactate + [2‐¹³C]pyruvate) to acetyl‐CoA production were measured both without and with 5.0 mM [U‐¹³C]3‐hydroxybutyrate in superfused rat hippocampal slices by ¹³C NMR non‐steady‐state isotopomer analysis of tissue glutamate and GABA. Without [U‐¹³C]3‐hydroxybutyrate, glucose, combined lactate + pyruvate, and unlabeled endogenous sources contributed (mean ± SEM) 70 ± 7%, 10 ± 2%, and 20 ± 8% of acetyl‐CoA, respectively. With [U‐¹³C]3‐hydroxybutyrate, glucose contributions significantly fell from 70 ± 7% to 21 ± 3% (p < 0.0001), combined lactate + pyruvate and endogenous contributions were unchanged, and [U‐¹³C]3‐hydroxybutyrate became the major acetyl‐CoA contributor (68 ± 3%) – about three‐times higher than glucose. A direct analysis of the GABA carbon 2 multiplet revealed that [U‐¹³C]3‐hydroxybutyrate contributed approximately the same acetyl‐CoA fraction as glucose, indicating that it was less avidly oxidized by GABAergic than glutamatergic neurons. The appearance of superfusate lactate derived from glycolysis of [1‐¹³C]glucose did not decrease significantly in the presence of 3‐hydroxybutyrate, hence total glycolytic flux (Krebs cycle inflow + exogenous lactate formation) was attenuated by 3‐hydroxybutyrate. This indicates that, under these conditions, 3‐hydroxybutyrate inhibited glycolytic flux upstream of pyruvate kinase. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous MR quantification of hepatic fat content,fatty acid composition,transverse relaxation time and magnetic susceptibility for the diagnosis of non‐alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) is characterized at histology by steatosis, hepatocyte ballooning and inflammatory infiltrates, with or without fibrosis. Although diamagnetic material in fibrosis and inflammation can be detected with quantitative susceptibility imaging, fatty acid composition changes in NASH relative to simple steatosis have also been reported. Therefore, our aim was to develop a single magnetic resonance (MR) acquisition and post‐processing scheme for the diagnosis of steatohepatitis by the simultaneous quantification of hepatic fat content, fatty acid composition, T ₂* transverse relaxation time and magnetic susceptibility in patients with non‐alcoholic fatty liver disease. MR acquisition was performed at 3.0 T using a three‐dimensional, multi‐echo, spoiled gradient echo sequence. Phase images were unwrapped to compute the B ₀ field inhomogeneity (ΔB ₀) map. The ΔB ₀‐demodulated real part images were used for fat–water separation, T ₂* and fatty acid composition quantification. The external and internal fields were separated with the projection onto dipole field method. Susceptibility maps were obtained after dipole inversion from the internal field map with single‐orientation Bayesian regularization including spatial priors. Method validation was performed in 32 patients with biopsy‐proven, non‐alcoholic fatty liver disease from which 12 had simple steatosis and 20 NASH. Liver fat fraction and T ₂* did not change significantly between patients with simple steatosis and NASH. In contrast, the saturated fatty acid fraction increased in patients with NASH relative to patients with simple steatosis (48 ± 2% versus 44 ± 4%; p  < 0.05) and the magnetic susceptibility decreased (?0.30 ± 0.27 ppm versus 0.10 ± 0.14 ppm; p  < 0.001). The area under the receiver operating characteristic curve for magnetic susceptibility as NASH marker was 0.91 (95% CI: 0.79–1.0). Simultaneous MR quantification of fat content, fatty acid composition, T ₂* and magnetic susceptibility is feasible in the liver. Our preliminary results suggest that quantitative susceptibility imaging has a high diagnostic performance for the diagnosis of NASH.     

Hyperpolarized [1,4‐13C]‐diethylsuccinate: a potential DNP substrate for in vivo metabolic imaging

The tricarboxylic acid (TCA) cycle performs an essential role in the regulation of energy and metabolism, and deficiencies in this pathway are commonly correlated with various diseases. However, the development of non‐invasive techniques for the assessment of the cycle in vivo has remained challenging. In this work, the applicability of a novel imaging agent, [1,4‐¹³C]‐diethylsuccinate, for hyperpolarized ¹³C metabolic imaging of the TCA cycle was explored. In vivo spectroscopic studies were conducted in conjunction with in vitro analyses to determine the metabolic fate of the imaging agent. Contrary to previous reports (Zacharias NM et al. J. Am. Chem. Soc. 2012; 134: 934–943), [¹³C]‐labeled diethylsuccinate was primarily metabolized to succinate‐derived products not originating from TCA cycle metabolism. These results illustrate potential issues of utilizing dialkyl ester analogs of TCA cycle intermediates as molecular probes for hyperpolarized ¹³C metabolic imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo MRSI of hyperpolarized [1-(13)C]pyruvate metabolism in rat hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than 1 year following diagnosis. Most patients with HCC are diagnosed at an advanced stage, and no efficient marker exists for the prediction of prognosis and/or response(s) to therapy. We have reported previously a high level of [1‐¹³C]alanine in an orthotopic HCC using single‐voxel hyperpolarized [1‐¹³C]pyruvate MRS. In the present study, we implemented a three‐dimensional MRSI sequence to investigate this potential hallmark of cellular metabolism in rat livers bearing HCC (n = 7 buffalo rats). In addition, quantitative real‐time polymerase chain reaction was used to determine the mRNA levels of lactate dehydrogenase A, nicotinamide adenine (phosphate) dinucleotide dehydrogenase quinone 1 and alanine transaminase. The enzyme levels were significantly higher in tumor than in normal liver tissues within each rat, and were associated with the in vivo MRSI signal of [1‐¹³C]alanine and [1‐¹³C]lactate after a bolus intravenous injection of [1‐¹³C]pyruvate. Histopathological analysis of these tumors confirmed the successful growth of HCC as a nodule in buffalo rat livers, revealing malignancy and hypervascular architecture. More importantly, the results demonstrated that the metabolic fate of [1‐¹³C]pyruvate conversion to [1‐¹³C]alanine significantly superseded that of [1‐¹³C]pyruvate conversion to [1‐¹³C]lactate, potentially serving as a marker of HCC tumors. Copyright © 2010 John Wiley & Sons, Ltd.     

Enhancing the [13C]bicarbonate signal in cardiac hyperpolarized [1‐13C]pyruvate MRS studies by infusion of glucose,insulin and potassium

A change in myocardial metabolism is a known effect of several diseases. MRS with hyperpolarized ¹³C‐labelled pyruvate is a technique capable of detecting changes in myocardial pyruvate metabolism, and has proven to be useful for the evaluation of myocardial ischaemia in vivo. However, during fasting, the myocardial glucose oxidation is low and the fatty acid oxidation (β‐oxidation) is high, which complicates the interpretation of pyruvate metabolism with the technique. The aim of this study was to investigate whether the infusion of glucose, insulin and potassium (GIK) could increase the myocardial glucose oxidation in the citric acid cycle, reflected as an increase in the [¹³C]bicarbonate signal in cardiac hyperpolarized [1‐¹³C]pyruvate MRS measurements in fasted rats. Two groups of rats were infused with two different doses of GIK and investigated by MRS after injection of hyperpolarized [1‐¹³C]pyruvate. No [¹³C]bicarbonate signal could be detected in the fasted state. However, a significant increase in the [¹³C]bicarbonate signal was observed by the infusion of a high dose of GIK. This study demonstrates that a high [¹³C]bicarbonate signal can be achieved by GIK infusion in fasted rats. The increased [¹³C]bicarbonate signal indicates an increased flux of pyruvate through the pyruvate dehydrogenase enzyme complex and an increase in myocardial glucose oxidation through the citric acid cycle. Copyright © 2013 John Wiley & Sons, Ltd.     

The effect of amiloride on biliary HCO-3 secretion in the anaesthetized pig

The effect of amiloride on biliary HCO^-₃-secretion in the anaesthetized pig. Acta Physiol Scand 130 , 447–455. Received 20 October 1986, accepted 23 February 1987. ISSN 0001–6772. Institute for Experimental Medical Research, University of Oslo and Surgical Department, Ullevaal Hospital, Oslo, Norway. The present study was performed on 29 anaesthetized pigs and shows that the bile acid ursodeoxycholic acid (UDCA) produces a flow of bile rich in HCO^-₃ compared with taurocholic acid (TCA). The slope relating biliary HCO^-₃ secretion to bile acid secretion was 0.59 (0.44–0.82) and 0.33 (0.29–0.38) during venous infusion of UDCA and TCA, respectively. We next wanted to evaluate the importance of Na⁺/H⁺ ion exchange for biliary HCO^-₃ secretion. High doses of amiloride were employed in order to impair the hepatic Na⁺/H⁺ ion exchanger. It was reasoned that any reduction in H⁺ efflux through the hepatic Na⁺/H⁺ ion exchanger involved in causing biliary HCO^-₃ secretion would be translated into an equimolar fall in biliary HCO^-₃ secretion. We found that amiloride (2.0 ± 10^--⁴ mol l^-1 plasma) reduced UDCA-dependent canalicular HCO^-₃ secretion by 26 (14–35)% without concurrently reducing bile acid secretion. Amiloride (2.9 ± 10^--⁴ mol 1^--¹ plasma) did not significantly reduce secretin-dependent ductular HCO^-₃ secretion. In this group of animals amiloride reduced bile acid secretion by 13 (5–22)%. It is concluded that Na⁺/H⁺ ion exchanger is essential for UDCA-dependent canalicular HCO^-₃ secretion, but not for secretin-dependent ductular HCO^-₃ secretion.     

Glucose metabolism via the pentose phosphate pathway, glycolysis and Krebs cycle in an orthotopic mouse model of human brain tumors

It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using orthotopic mouse models of human glioblastoma (GBM) and renal cell carcinoma metastatic to brain, we estimated the activity of the PPP relative to glycolysis by infusing [1,2‐¹³C₂]glucose. The [3‐¹³C]lactate/[2,3‐¹³C₂]lactate ratio was similar for both the GBM and brain metastasis and their respective surrounding brains (GBM, 0.197 ± 0.011 and 0.195 ± 0.033, respectively (p = 1); metastasis: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than the PPP flux in these tumors, and that the PPP flux into the lactate pool is similar in both tumors. Remarkably, ¹³C–¹³C coupling was observed in molecules derived from Krebs cycle intermediates in both tumor types, denoting glucose oxidation. In the renal cell carcinoma, in contrast with GBM, ¹³C multiplets of γ‐aminobutyric acid (GABA) differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. In addition, the orthotopic renal tumor, the patient's primary renal mass and brain metastasis were all strongly immunopositive for the 67‐kDa isoform of glutamate decarboxylase, as were 84% of tumors on a renal cell carcinoma tissue microarray of the same histology, suggesting that GABA synthesis is cell autonomous in at least a subset of renal cell carcinomas. Taken together, these data demonstrate that ¹³C‐labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Growth temperature associated protein expression and membrane fatty acid composition profiles of Salmonella enterica serovar Typhimurium

Total cellular proteins and fatty acid composition profiles of mid‐log phase cells of Salmonella enterica serovar Typhimurium grown at 8, 25, 37 or 42 °C were separated by 2D‐PAGE and FAME analysis. Growth temperature associated protein expression can be grouped into 3 thermal classes which include proteins whose expression is: I) optimal at 37 °C, meaning their expression peaked at 37 °C; II) up‐regulated with an increase in growth temperature; III) down‐regulated with increase in growth temperature; meaning their expression peaked at 8 °C. At higher growth temperatures, proteins belonging to the functional groups of amino acid transport and metabolism, nucleotide metabolism, energy metabolism and post‐translation modifications (chaperones) are present in substantially higher amounts. This increase in abundance is regulated in a temperature dependent manner. It is important to point out that proteins involved in energy metabolism observed in higher amounts at higher growth temperatures all belong to the glycolysis pathway, while at 8 °C they belonged to the TCA cycle. Increase in growth temperatures results in a decrease in membrane fatty acid unsaturation and an increase in saturated and cyclic fatty acids. These results provide an insight into the dynamic molecular and physiological responses of Salmonella Typhimurium during growth at different temperatures. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Plasma cholecystokinin levels in Prader‐Willi syndrome and obese subjects

The cardinal feature of individuals with Prader‐Willi syndrome (PWS) is severe hyperphagia‐mediated obesity resulting from a faulty satiety mechanism. PWS is the most common genetic cause of marked obesity. Cholecystokinin (CCK) is a 33‐amino‐acid peptide found in high levels in the gut and brain involved in mediating the satiety response to meals. Free fatty acids (FFA) are responsible for the stimulation of CCK release after a fatty meal, and CCK and plasma FFA levels rise in tandem in normal individuals. Fasting plasma CCK levels were measured by radio‐immunoassay in 33 PWS subjects with a mean age of 22.2 years ± 8.1 years and 24 obese control subjects without a known cause of their obesity with a mean age of 28.7 years ± 12.9 years. Consistent with previous findings, neither fasting plasma FFA levels (617.5 versus 486.8 μm/mL) or CCK levels (21.0 versus 19.1 pg/mL) were significantly different in PWS or control subjects, respectively. However, there was a significant correlation between fasting plasma FFA and CCK levels in obese subjects (r = 0.64, P < 0.01), this correlation was completely lacking in PWS subjects (r = −0.06, P = 0.79). This difference in correlation coefficients constitutes a large effect. There were no significant effects observed for genetic subtypes (15q11‐q13 deletion or maternal disomy 15), body mass index, percentage of fat, plasma levels of insulin, C‐peptide, glucagon or leptin, age, or gender on CCK levels in our PWS subjects. These results suggest that differences in the peripheral CCK response to FFA levels may be a factor contributing to the altered satiety response in PWS subjects. Am. J. Med. Genet. 95:67–70, 2000. © 2000 Wiley‐Liss, Inc.     

In vivo investigation of cardiac metabolism in the rat using MRS of hyperpolarized [1‐13C] and [2‐13C]pyruvate

Hyperpolarized ¹³C MRS allows the in vivo assessment of pyruvate dehydrogenase complex (PDC) flux, which converts pyruvate to acetyl‐coenzyme A (acetyl‐CoA). [1‐¹³C]pyruvate has been used to measure changes in cardiac PDC flux, with demonstrated increase in ¹³C‐bicarbonate production after dichloroacetate (DCA) administration. With [1‐¹³C]pyruvate, the ¹³C label is released as ¹³CO₂/¹³C‐bicarbonate, and, hence, does not allow us to follow the fate of acetyl‐CoA. Pyruvate labeled in the C₂ position has been used to track the ¹³C label into the TCA (tricarboxylic acid) cycle and measure [5‐¹³C]glutamate as well as study changes in [1‐¹³C]acetylcarnitine with DCA and dobutamine. This work investigates changes in the metabolic fate of acetyl‐CoA in response to metabolic interventions of DCA‐induced increased PDC flux in the fed and fasted state, and increased cardiac workload with dobutamine in vivo in rat heart at two different pyruvate doses. DCA led to a modest increase in the ¹³C labeling of [5‐¹³C]glutamate, and a considerable increase in [1‐¹³C]acetylcarnitine and [1,3‐¹³C]acetoacetate peaks. Dobutamine resulted in an increased labeling of [2‐¹³C]lactate, [2‐¹³C]alanine and [5‐¹³C]glutamate. The change in glutamate with dobutamine was observed using a high pyruvate dose but not with a low dose. The relative changes in the different metabolic products provide information about the relationship between PDC‐mediated oxidation of pyruvate and its subsequent incorporation into the TCA cycle compared with other metabolic pathways. Using a high dose of pyruvate may provide an improved ability to observe changes in glutamate. Copyright © 2013 John Wiley & Sons, Ltd.     

Reducing plasma free fatty acids by acipimox improves glucose tolerance in high‐fat fed mice

To study whether free fatty acids (FFAs) contribute to glucose intolerance in high‐fat fed mice, the derivative of nicotinic acid, acipimox, which inhibits lipolysis, was administered intraperitoneally (50 mg kg^?1) to C57BL/6J mice which had been on a high‐fat diet for 3 months. Four hours after administration of acipimox, plasma FFA levels were reduced to 0.46 ± 0.06 mmol L^?1 compared with 0.88 ± 0.10 mmol L^?1 in controls (P < 0.001). At this point, the glucose elimination rate after an intravenous glucose load (1 g kg^?1) was markedly improved. Thus, the elimination constant (K_G) for the glucose disposal between 1 and 50 min after the glucose challenge was increased from 0.54 ± 0.01% min^?1 in controls to 0.66 ± 0.01% min^?1 by acipimox (P < 0.001). In contrast, the acute insulin response to glucose (1–5 min) was not significantly different between the groups, although the area under the insulin for the entire 50‐min period after glucose administration was significantly reduced by acipimox from 32.1 ± 2.9 to 23.9 ± 1.2 nmol L^?1 × 50 min (P=0.036). This, however, was mainly because of lower insulin levels at 20 and 50 min because of the lowered glucose levels. In contrast, administration of acipimox to mice fed a normal diet did not affect plasma levels of FFA or the glucose elimination or insulin levels after the glucose load. It is concluded that reducing FFA levels by acipimox in glucose intolerant high‐fat fed mice improves glucose tolerance mainly by improving insulin sensitivity making the ambient islet function adequate, suggesting that increased FFA levels are of pathophysiological importance in this model of glucose intolerance.