In vivo quantification of neuro-glial metabolism and glial glutamate concentration using 1H-[13C] MRS at 14.1T.

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo (13) C NMR spectroscopy coupled with the infusion labeled glial-specific substrate, such as acetate. In this study, we infused alpha-chloralose anesthetized rats with [2-(13) C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using (1) H-[(13) C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two-compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (Vg ) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (VNT ) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (VPC ) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (Vx (g) and Vx (n) ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations. In this (13) C-acetate labeling study, we propose a refined two-compartment analysis of brain energy metabolism based on (13) C turnover curves of acetate, glutamate and glutamine measured with state of the art in vivo dynamic MRS at high magnetic field in rats, enabling a deeper understanding of the specific role of glial cells in brain oxidative metabolism. In addition, the robustness of the metabolic fluxes determination relative to MRS data quality was carefully studied.

Energy cost of glucose storage in human subjects during glucose-insulin infusions.

The effect of graded levels of hyperinsulinemia on energy expenditure, while euglycemia was maintained by glucose infusion, was examined in 22 healthy young male volunteers by using the euglycemic insulin clamp technique in combination with indirect calorimetry. Insulin was infused at five rates to achieve steady-state hyperinsulinemic plateaus of 62 +/- 4, 103 +/- 5, 170 +/- 10, 423 +/- 16, and 1,132 +/- 47 microU/ml. Total body glucose uptake during each of the five insulin clamp studies was 0.41, 0.50, 0.66, 0.74, and 0.77 g/min, respectively. Glucose storage (calculated from the difference between total body glucose uptake minus total glucose oxidation) was 0.25, 0.29, 0.43, 0.49, and 0.52 g/min for each group, respectively, and represented over 60-70% of total glucose uptake. The net increment in energy expenditure after intravenous glucose was 0.08, 0.10, 0.14, 0.17, and 0.23 kcal/min, respectively. Throughout the physiological and supraphysiological range of insulinemia, there was a significant relationship (r = 0.95, P less than 0.001) between the increment in energy expenditure and glucose storage, indicating an energy cost of 0.45 kcal/g glucose stored. However, at each level of hyperinsulinemia, the theoretical value for the energy cost of glucose storage (assuming that all of the glucose is stored in the form of glycogen) could account for only 45-63% of the actual increase in energy expenditure that was measured by indirect calorimetry. These results indicate that factors in addition to glucose storage as glycogen must be responsible for the increase in energy expenditure that accompanies glucose infusion.

Effect of carbohydrate overfeeding on whole body and adipose tissue metabolism in humans.

OBJECTIVE: To evaluate the effect of a 4-day carbohydrate overfeeding on whole body net de novo lipogenesis and on markers of de novo lipogenesis in subcutaneous adipose tissue of healthy lean humans. RESEARCH METHODS AND PROCEDURES: Nine healthy lean volunteers (five men and four women) were studied after 4 days of either isocaloric feeding or carbohydrate overfeeding. On each occasion, they underwent a metabolic study during which their energy expenditure and net substrate oxidation rates (indirect calorimetry), and the fractional activity of the pentose-phosphate pathway in subcutaneous adipose tissue (subcutaneous microdialysis with 1,6(13)C2,6,6(2)H2 glucose) were assessed before and after administration of glucose. Adipose tissue biopsies were obtained at the end of the experiments to monitor mRNAs of key lipogenic enzymes. RESULTS: Carbohydrate overfeeding increased basal and postglucose energy expenditure and net carbohydrate oxidation. Whole body net de novo lipogenesis after glucose loading was markedly increased at the expense of glycogen synthesis. Carbohydrate overfeeding also increased mRNA levels for the key lipogenic enzymes sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase. The fractional activity of adipose tissue pentose-phosphate pathway was 17% to 22% and was not altered by carbohydrate overfeeding. DISCUSSION: Carbohydrate overfeeding markedly increased net de novo lipogenesis at the expense of glycogen synthesis. An increase in mRNAs coding for key lipogenic enzymes suggests that de novo lipogenesis occurred, at least in part, in adipose tissue. The pentose-phosphate pathway is active in adipose tissue of healthy humans, consistent with an active role of this tissue in de novo lipogenesis.

Effects of dexamethasone on hepatic glucose production and fructose metabolism in healthy humans.

This study was designed to determine whether glucocorticoids alter autoregulation of glucose production and fructose metabolism. Two protocols with either dexamethasone (DEX) or placebo (Placebo) were performed in six healthy men during hourly ingestion of[13C]fructose (1.33 mmol.kg-1.h-1) for 3 h. In both protocols, endogenous glucose production (EGP) increased by 8 (Placebo) and 7% (DEX) after fructose, whereas gluconeogenesis from fructose represented 82 (Placebo) and 72% (DEX) of EGP. Fructose oxidation measured from breath 13CO2 was similar in both protocols [9.3 +/- 0.7 (Placebo) and 9.6 +/- 0.5 mumol.kg-1.min-1 (DEX)]. Nonoxidative carbohydrate disposal, calculated as fructose administration rate minus net carbohydrate oxidation rate after fructose ingestion measured by indirect calorimetry, was also similar in both protocols [5.8 +/- 0.8 (Placebo) and 5.9 +/- 2.0 mumol.kg-1.min-1 (DEX)]. We concluded that dexamethasone 1) does not alter the autoregulatory process that prevents a fructose-induced increase in gluconeogenesis from increasing total glucose production and 2) does not affect oxidative and nonoxidative pathways of fructose. This indicates that the insulin-regulated enzymes involved in these pathways are not affected in a major way by dexamethasone.

Postischemic recovery of heart metabolism and function: role of mitochondrial fatty acid transfer.

Postischemic recovery of contractile function is better in hearts from fasted rats than in hearts from fed rats. In this study, we examined whether feeding-induced inhibition of palmitate oxidation at the level of carnitine palmitoyl transferase I is involved in the mechanism underlying impaired recovery of contractile function. Hearts isolated from fasted or fed rats were submitted to no-flow ischemia followed by reperfusion with buffer containing 8 mM glucose and either 0.4 mM palmitate or 0.8 mM octanoate. During reperfusion, oxidation of palmitate was higher after fasting than after feeding, whereas oxidation of octanoate was not influenced by the nutritional state. In the presence of palmitate, recovery of left ventricular developed pressure was better in hearts from fasted rats. Substitution of octanoate for palmitate during reperfusion enhanced recovery of left ventricular developed pressure in hearts from fed rats. However, the chain length of the fatty acid did not influence diastolic contracture. The results suggest that nutritional variation of mitochondrial fatty acid transfer may influence postischemic recovery of contractile function.

Detection of neuronal activity and metabolism in a model of dehydration-induced anorexia in rats at 14.1 T using manganese-enhanced MRI and 1H MRS.

In this study, hypothalamic activation was performed by dehydration-induced anorexia (DIA) and overnight food suppression (OFS) in female rats. The assessment of the hypothalamic response to these challenges by manganese-enhanced MRI showed increased neuronal activity in the paraventricular nuclei (PVN) and lateral hypothalamus (LH), both known to be areas involved in the regulation of food intake. The effects of DIA and OFS were compared by generating T-score maps. Increased neuronal activation was detected in the PVN and LH of DIA rats relative to OFS rats. In addition, the neurochemical profile of the PVN and LH were measured by (1) H MRS at 14.1T. Significant increases in metabolite levels were measured in DIA and OFS relative to control rats. Statistically significant increases in γ-aminobutyric acid were found in DIA (p=0.0007) and OFS (p<0.001) relative to control rats. Lactate increased significantly in DIA (p=0.03), but not in OFS, rats. This work shows that manganese-enhanced MRI coupled to (1) H MRS at high field is a promising noninvasive method for the investigation of the neural pathways and mechanisms involved in the control of food intake, in the autonomic and endocrine control of energy metabolism and in the regulation of body weight.

Energy-nitrogen balances and protein turnover in small and appropriate for gestational age low birthweight infants.

The aim of the present study was to compare, under the same nursing conditions, the energy-nitrogen balance and the protein turnover in small for gestational age (SGA) and appropriate for gestational age (AGA) low birthweight infants. We compared 8 SGA's (mean +/- s.d.: gestational age 35 +/- 2 weeks, birthweight 1520 +/- 330 g) to 11 AGA premature infants (32 +/- 2 weeks, birthweight 1560 +/- 240 g). When their rate of weight gain was above 15 g/kg/d (17.6 +/- 3.0 and 18.2 +/- 2.6 g/kg/d, mean postnatal age 18 +/- 10 and 20 +/- 9 d respectively) they were studied with respect to their metabolizable energy intake, their energy expenditure, their energy and protein gain and their protein turnover. Energy balance was assessed by the difference between metabolizable energy and energy expenditure as measured by indirect calorimetry. Protein gain was calculated from the amount of retained nitrogen. Protein turnover was estimated by a stable isotope enrichment technique using repeated nasogastric administration of 15N-glycine for 72 h. Although there was no difference in their metabolizable energy intakes (110 +/- 12 versus 108 +/- 11 kcal/kg/d), SGA's had a higher rate of resting energy expenditure (64 +/- 8 versus 57 +/- 8 kcal/kg/d, P less than 0.05). Protein gain and composition of weight gain was very similar in both groups (2.0 +/- 0.4 versus 2.1 +/- 0.4 g protein/kg/d; 3.5 +/- 1.1 versus 3.3 +/- 1.4 g fat/kg/d in SGA's and AGA's respectively). However, the rate of protein synthesis was significantly lower in SGA's (7.7 +/- 1.6 g/kg/d) as compared to AGA's (9.7 +/- 2.8 g/kg/d; P less than 0.05). It is concluded that SGA's have a more efficient protein gain/protein synthesis ratio since for the same weight and protein gains, SGA's show a 20 per cent slower protein turnover. They might therefore tolerate slightly higher protein intakes. Postconceptional age seems to be an important factor in the regulation of protein turnover.

Energy expenditure in obese women before and during weight loss, after refeeding, and in the weight-relapse period.

In 10 moderately obese women, 24-h energy expenditure (24EE) was measured in a respiration chamber under four conditions: 1) before weight loss (body weight = 77.9 kg), 2) during weight loss (63.9 kg), 3) after realimentation (62.5 kg), and 4) 6-15 mo after the study diet with ad libitum diet (67.7 kg). The 14 +/- 8 kg (mean +/- SD) weight loss produced a decrease in 24EE of 1498 +/- 1138 kJ/d (P < 0.001), ie, a decrease of weight of 107 kJ.kg body wt-1.d-1. The subsequent 24EE (conditions 3 and 4) remained lower than the value before weight loss. A significant correlation was found between changes before and after weight regain in basal respiratory quotient (RQ) and the spontaneous rate of body-weight gain after cessation of the period of low energy intake (r = 0.89, P < 0.01); this suggests that the value of the postabsorptive RQ may be a predictor of relapse of weight gain. After discontinuation of the low energy diet, an elevated postabsorptive RQ shows that the endogenous lipid oxidation is low, a condition favoring weight gain.

Free-living energy expenditure assessed by two different methods in rural Gambian men.

OBJECTIVE: To assess total free-living energy expenditure (EE) in Gambian farmers with two independent methods, and to determine the most realistic free-living EE and physical activity in order to establish energy requirements for rural populations in developing countries. DESIGN: In this cross-sectional study two methods were applied at the same time. SETTING: Three rural villages and Dunn Nutrition Centre Keneba, MRC, The Gambia. SUBJECTS: Eight healthy, male subjects were recruited from three rural Gambian villages in the sub-Sahelian area (age: 25 +/- 4y; weight: 61.2 +/- 10.1 kg; height: 169.5 +/- 6.5 cm, body mass index: 21.2 +/- 2.5 kg/m2). INTERVENTION: We assessed free-living EE with two inconspicuous and independent methods: the first one used doubly labeled water (DLW) (2H2 18O) over a period of 12 days, whereas the second one was based on continuous heart rate (HR) measurements on two to three days using individual regression lines (HR vs EE) established by indirect calorimetry in a respiration chamber. Isotopic dilution of deuterium (2H2O) was also used to assess total body water and hence fat-free mass (FFM). RESULTS: EE assessed by DLW was found to be 3880 +/- 994 kcal/day (16.2 +/- 4.2 MJ/day). Expressed per unit body weight the EE averaged 64.2 +/- 9.3 kcal/kg/d (269 +/- 38 kJ/kg/d). These results were consistent with the EE results assessed by HR: 3847 +/- 605 kcal/d (16.1 +/- 2.5 MJ/d) or 63.4 +/- 8.2 kcal/kg/d (265 +/- 34kJ/kg/d). Physical activity index, expressed as a multiple of basal metabolic rate (BMR), averaged 2.40 +/- 0.41 (DLW) or 2.40 +/- 0.28 (HR). CONCLUSIONS: These findings suggest an extremely high level of physical activity in Gambian men during intense agricultural work (wet season). This contrasts with the relative food shortage, previously reported during the harvesting period. We conclude that the assessment of EE during the agricultural season in non-industrialized countries needs further investigations in order to obtain information on the energy requirement of these populations. For this purpose the use of the DLW and HR methods have been shown to be useful and complementary.

Energy expenditure during walking and running in obese and nonobese prepubertal children.

We measured body composition and energy expenditure during walking and running on a treadmill in 40 prepubertal children: 23 obese children (9.3 +/- 1.1 years of age; 46 +/- 10 kg (mean +/- SD)) and 17 nonobese matched control children (9.2 +/- 0.6 years of age; 30 +/- 5 kg). Energy expenditure was assessed by indirect calorimetry with a standard open-circuit method. At the same speed of exercise, the energy expenditure was significantly (p < 0.01) greater in obese than in control children, in both boys and girls. Expressed per kilogram of body weight or per kilogram of fat-free mass, the energy expenditure was comparable in the two groups. Obese children had a significantly (p < 0.01) larger pulmonary ventilatory response to exercise than did control children. Heart rate was comparable in boys and girls combined but significantly higher (p < 0.05) in obese subjects, if boys and girls were analyzed separately. These data indicate that walking and running are energetically more expensive for obese children than for children of normal body weight. The knowledge of these energy costs could be useful in devising a physical activity program to be used in the treatment of obese children.

Energy expenditure before and during energy restriction in obese patients.

Twenty-four hour energy expenditure (24 EE), resting metabolic rate (RMR), spontaneous physical activity and body composition were determined in 7 obese patients (5 females, 2 males, 174 +/- 9% IBW, 38 +/- 2% fat mass) on 2 different occasions: before weight reduction, and after 10 to 16 weeks on a hypocaloric diet as outpatients, the recommended energy intake varying from 3500 to 4700 kJ/day depending on the subject. Mean body weight loss was 12.6 +/- 1.9 kg, ie 13% of initial body weight, 72% being fat. Twenty-four hour energy expenditure (24 EE) was measured in a respiration chamber with all the subjects receiving 10418 kJ/d before weight reduction and an average of 3360 +/- 205 kJ/d while on the diet. When expressed in absolute values, both 24 EE and RMR decreased during the hypocaloric diet from 9819 +/- 442 to 8229 +/- 444 and from 7262 +/- 583 to 6591 +/- 547 kJ/d respectively. On the basis of fat-free-mass (FFM), 24 EE decreased from 168 +/- 6 to 148 +/- 5 kJ/kg FFM/d whereas RMR was unchanged (approximately 120 kJ/kg FFM/d). Approximately one half of the 24 EE reduction (1590 kJ/d) was accounted for by a decrease in RMR, the latter being mainly accounted for by a reduction in FFM. Most of the remaining decline in 24 EE can be explained by a decreased thermic effect of food, and by the reduced cost of physical activity mainly due to a lower body weight. Therefore, there seems little reason to evoke additional mechanisms to explain the decline in energy expenditure during dieting.

Unstable shoes increase energy expenditure of obese patients.

BACKGROUND: Ergonomic unstable shoes, which are widely available to the general population, could increase daily non-exercise activity thermogenesis as the result of increased muscular involvement. We compared the energy expenditure of obese patients during standing and walking with conventional flat-bottomed shoes versus unstable shoes. METHODS: Twenty-nine obese patients were asked to stand quietly and to walk at their preferred walking speed while wearing unstable or conventional shoes. The main outcome measures were metabolic rate of standing and gross and net energy cost of walking, as assessed with indirect calorimetry. RESULTS: Metabolic rate of standing was higher while wearing unstable shoes compared with conventional shoes (1.11 ± 0.20 W/kg(-1) vs 1.06 ± 0.23 W/kg(-1), P=.0098). Gross and net energy cost of walking were higher while wearing unstable shoes compared with conventional shoes (gross: 4.20 ± 0.42 J/kg(-1)/m(-1)vs 4.01 ± 0.39 J/kg(-1)/m(-1), P=.0035; net: 3.37 ± 0.41 J/kg(-1)/m(-1) vs 3.21 ± 0.37 J/kg(-1)/m(-1); P=.032). CONCLUSION: In obese patients, it is possible to increase energy expenditure of standing and walking by means of ergonomic unstable footwear. Long-term use of unstable shoes may eventually prevent a positive energy balance.