Myocardial energy metabolism in ischemic preconditioning and cardioplegia: A metabolic control analysis

For both, cardioplegia (CP) and ischemic preconditioning (IP), increased ischemic tolerance with reduction in infarct size is well documented. These cardioprotective effects are related to a limitation of high energy phosphate (HEP) depletion. As CP and IP have to be assumed to act by different mechanisms, their effects on myocardial HEP metabolism cannot be assumed to be identical. Therefore, a systematic analysis of myocardial HEP metabolism for both procedures and their combination was performed, addressing the question whether there are different effects on myocardial HEP metabolism by IP and CP. In this study, metabolic control analysis was used to analyze the regulation of HEP metabolism. In open chest pigs subjected to 45 min LAD occlusion (index ischemia), CP and IP preserved myocardial ATP (control (C) 0.14 ± 0.05 μmol/g wwt; CP: 0.95 ± 0.14, IP: 0.61 ± 0.12; p<0.05 C vs. CP and IP) and reduced myocardial necrosis (infarct size IA/RA: C: 90.0 ± 3.0%; CP: 0.0 ± 0.0% but patchy necroses; IP: 5.05 ± 2.1%; p<0.05 C vs. CP and IP). The effects on HEP metabolism, however, were different: CP acted predominantly by slowing down the breakdown of phosphocreatine (PCr) during early phases of ischemia (C: ΔPCr 0–2 min: 5.24 ± 0.32 μmol/g wwt; CP: ΔPCr 0–2 min: 3.38 ± 0.23 μmol/g wwt, p<0.05 vs. C), leaving ATP breakdown during later stages unaffected (C: ΔATP 5–45 min: 1.77 ± 0.11 μmol/g wwt CP: ΔATP 5–45 min: 1.59 ± 0.28 μmol/g wwt, n.s. vs. C). In contrast to CP, in IP PCr breakdown was even increased (IP: ΔPCr 0–2 min: 7.06 ± 0.34 μmol/g wwt, p<0.05 vs. C), but ATP depletion greatly attenuated (IP: ΔATP 5–45 min: 0.48 ± 0.10 μmol/g wwt, p<0.05 vs. C and CP). Combining IP and CP yielded an additive effect with slowing down the breakdown of both PCr (IP+CP: ΔPCr 0–2 min: 5.09± 0.35 μmol/g wwt, p<0.05 vs. C and IP) and ATP (IP+CP: ΔATP 5–45 min: 0.56 ± 0.48 μmol/g wwt, p<0.05 vs. C and CP), resulting in a higher ATP content at the end of index ischemia (1.86 ± 0.46 μmol/g wwt, p<0.05 vs. C, CP and IP). Compared to IP, combining IP+CP achieved also a further reduction in infarct size (IA/RA: 0.0 ± 0.0%, p<0.05 vs IP) and—compared to CP—a disappearance of the patchy necroses. {The concept of major differences in myocardial HEP metabolism during CP and IP is further supported at a molecular level by metabolic control analysis. CP but not IP slowed down the CK reaction velocity at high PCr levels. In contrast to CP exerting a continuous decline in vATPase for any given ATP level, in IP myocardium ATPase reaction velocity was even increased at higher ATP contents, whereas a marked decrease in ATPase reaction velocity was found if ATP levels decreased. The equilibrium of the CK-reaction remained unchanged following CP, whereas IP induced a changing CK equilibrium, which was the more shifted towards PCr the more myocardial HEP content decreased. The data demonstrate different effects of CP and IP on myocardial HEP metabolism, i.e. PCr and ATP breakdown as well as the apparent equilibrium of the creatine kinase (CK)-reaction. For these reasons the combination of the two protective interventions has an additive effect. (Mol Cell Biochem 278: 222–232, 2005)     

Kinetics of Transport and Phosphorylation of 2-Fluoro-2-Deoxy-d-Glucose in Rat Brain

Abstract: The kinetics of transport across the blood-brain barrier and metabolism in brain (hemisphere) of [¹⁴C]2-fluoro-2-deoxy-d -glucose (FDG) were compared to that of [³H]2-deoxy-d -glucose (DG) and d -glucose in the pentobarbital-anesthetized adult rat. Saturation kinetics of transport were measured with the brain uptake index (BUI) method. The BUI for FDG was 54.3 ± 5.6. Nonlinear regression analysis gave a K_m of 6.9 ± 1.1 mM and a V_max of 1.70 ± 0.32 μmol/min/g. The K₁ for glucose inhibition of FDG transport was 10.7 ± 4.4 mM. The kinetic constants of influx (k₁) and efflux (K₂) for FDG were calculated from the K_m, V_max, and glucose concentrations of the hemisphere and plasma (2.3 ± 0.2 μmol/g and 9.9 ± 0.4 mM, respectively). The transport coefficient (k_{1 FDG}/k₁glucose) was 1.67 ± 0.07 and the phosphorylation constant was 0.55 ± 0.16. The predicted lumped constant for FDG was 0.89, whereas the measured hexose utilization index for FDG was 0.85 ± 0.16. Conclusion: The value for the lumped constant can be predicted on the basis of the known kinetic constants of FDG and glucose transport and metabolism, as well as brain and plasma glucose levels. Knowledge of the lumped constant is crucial in interpreting data obtained from ¹⁸FDG analysis of regional glucose utilization in human brain in pathological states. We propose that the lumped constant will rise to a maximum equal to the transport coefficient for FDG under conditions of transport limitation (hypoglycemia) or elevated glycolysis (ischemia, seizures), and will fall to a minimum equal to the phosphorylation coefficient during phosphorylation limitation (extreme hyperglycemia).     

Determination of vitamins D,A, and E in sera and vitamin D in milk from captive and free-ranging polar bears (Ursus maritimus), and 7-dehydrocholesterol levels in skin from captive polar bears

The difference between serum levels from 36 captive and 56 free-ranging polar bears (Ursus maritimus) for 25-hydroxyvitamin D (25-OH-D) was found not to be significant (mean ± SD = 348 ± 215 nmol/L [captive], 360 ± 135 nmol/L [free-ranging], t = 0.30, df = 52.8, P = 0.76), whereas the difference for retinol and α-tocopherol was significant (retinol, 1.37 ± 0.67 μmol/L [captive] 1.89 ± 0.63 μmol/L [free-ranging], t = 3.88, df = 72.4, P <0.001, α-tocopherol, 18.56 ± 18.56 μmol/L [captive], 48.76 ± 13.92 μmol/L [free-ranging], t = 7.85, df = 61.9, P < 0.001). Due to the high fat content in the polar bear diet, seal blubber may be the source of these fat-soluble vitamins. Six skin biopsies were analyzed from captive polar bears at the Denver Zoological Gardens for 7-dehydrocholesterol levels and found to contain 0.11 ± 0.03 nmol/cm². This finding also helps to support the contention that the source of vitamin D for polar bears may be ingestion and not cutaneous production. Vitamin D content in the milk from one captive sow in the den (0.14 nmol/g) and 10 free-ranging sows with cubs of the year out on the ice pack (0.0042 ± 0.0073 nmol/g) were also evaluated. It would be helpful to evaluate additional milk samples from denning and non-denning sows with cubs to see whether vitamin D content varies according to the stage of lactation. Zoo Biol 17:285–293, 1998. © 1998 Wiley- Liss, Inc.     

Lactate Uptake and Release in the Presence of Glucose by Sympathetic Ganglia of Chicken Embryos and by Neuronal and Nonneuronal Cultures Prepared from These Ganglia

Abstract: Uptake and output of lactate were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14–15 days old. The chains, typically containing 30–40 μg of protein, were incubated in Eagle's minimum essential medium containing bicarbonate buffer, 6–17 mM glucose, various concentrations of lactate, and either [U-¹⁴C]lactate, [1-¹⁴C]glucose, or [6-¹⁴C]glucose. The average rate of uptake of labeled lactate was measured with incubations of 5–6 h, starting with various external lactate concentrations. From these data the instantaneous relation between lactate uptake rate and concentration was deduced with a simple computerized model. The instantaneous uptake rate increased with the concentration according to a relation that fit the Michaelis-Menten equation, with V_max = 360 μmol/g protein/h and K_m = 4.8 mM. Substantial fractions of the lactate carbon were recovered from tissue constituents and in several nonvolatile products in the medium, as well as in CO₂. Glucose uptake averaged about 108 μmol/g protein/h and did not vary greatly with external lactate concentration, although the metabolic partitioning of glucose carbon was considerably affected. Regardless of initial concentration, the lactate concentration in the medium tended to change towards approximately 0.6 mM, showing that uptake equaled output at this level, with rates at about 40 μmol/g protein/h. With the steady-state concentration of 0.6 mM lactate, about 20% of the glucose carbon was shunted out into the medium before it was reabsorbed and metabolized into various products. Lactate uptakes by neuronal and nonneuronal cultures prepared from the ganglia did not differ consistently from one another or from uptake by undissociated ganglia. The neuronal cultures tended to oxidize a greater fraction of the consumed lactate to CO₂ and to convert a smaller fraction of the lactate to products in the medium than did the nonneuronal cultures. Computer modeling, using known parameters for blood-brain transport of lactate in the adult rat and data on uptake by the ganglia, suggests that lactate may supply substantial fuel to the brain, even in the presence of abundant glucose, when the lactate concentration in the blood is raised to levels commonly observed in exercising humans, such as 10–20 mM. This is in agreement with the findings of several investigators in hypoglycemic humans and in animals with intermediate blood lactate concentrations.     

Neither Moderate Hypoxia nor Mild Hypoglycaemia Alone Causes Any Significant Increase in Cerebral [Ca2±i: Only a Combination of the Two Insults Has This Effect.: A 31P and 19F NMR Study

(1) The energy state and free intracellular calcium concentration ([Ca^2±_i) of super-fused cortical slices were measured in moderate hypoxia (～65 μM O₂), in mild hypoglycaemia (0.5 mM glucose), and in combinations of the two insults using ¹⁹F and ³¹P NMR spectroscopy. (2) Neither hypoxia nor hypoglycaemia alone caused any significant change in [Ca^2±_i. Hypoxia caused a 40% fall in phosphocreatine (PCr) content but not in ATP level, and hypoglycaemia produced a slight fall in both (as expected from previous studies). These changes in the energy state recovered on return to control conditions. (3) A combined sequential insult (hypoxia, followed by hypoxia plus hypoglycaemia) produced a 100% increase in [Ca^2±, and a decrease in PCr level to ～25% of control. The reverse combined sequential insult (hypoglycaemia, followed by hypoglycaemia plus hypoxia) had the same effect. On return to control conditions there was some decrease in [Ca^2±_i and a small increase in PCr content, but neither recovered to control levels. (4) Exposure of the tissue to the combined simultaneous insult (hypoxia plus hypoglycaemia) immediately after the control spectra had been recorded resulted in a fivefold increase in [Ca^2±_i and a similar decrease in PCr level to 20–25% of control. There was little if any change of [Ca^2±_i or PCr level on return to control conditions. (5) These results are discussed in terms of metabolic adaptation of some but not all of the cortical cells to the single type of insult, which renders the tissues less vulnerable to the combined insult.     

Metabolic recovery in herring larvae following strenuous activity

Larvae of spring spawning Clyde herring Clupea harengus L. were reared at 5 and 12° C. Metabolism following burst swimming was studied in 7-day-old larvae at their respective rearing temperatures. Escape responses were repeatedly elicited using tactile stimulation for a period of 3 min. Larval herring were hard to fatigue and still responded to tactile stimuli after 3 min. Whole larvae were freeze-quenched in liquid nitrogen, either immediately after exercise, or after periods of recovery of up to 24 h. Samples were freeze-dried and analysed for whole body creatine (Cr), phosphocreatine (PCr), ATP, ADP, AMP, lactate, glucose, and glycogen using high performance liquid chromatography and enzymatic methods. The exercise regime resulted in a marked decrease in PCr, ATP and glycogen concentrations and an increase in creatine, glucose and lactate concentrations whereas there was no significant change in either AMP or ADP concentrations. The extent of phosphagen hydrolysis (approx. 110 to 15μmol PCr g ⁻¹ dry body mass) and lactate accumulation (approx. 7 to 40 μmol lactate g⁻¹ dry body mass) over the exercise period was similar at the two temperatures, consistent with a relatively constant degree of effort. The rates of recovery of PCr and ATP were essentially the same at 5 and 12° C; returning to resting levels after approximately 30 min. Lactate and glycogen concentrations were restored 60 min after exercise at both temperatures. Maximum lactate clearance rates (1.2 μmol min ⁻¹ g ⁻¹ wet muscle mass) were an order of magnitude faster than reported for adult fish in the literature.     

Stimulation of ATP synthesis in hypothermically perfused dog kidneys by adenosine and PO4

During continuous hypothermic perfusion of dog kidneys there occurs a gradual decrease in ATP from about 1.4 to 0.6 μmol/g wet wt after 5 days of preservation. The loss of ATP can be prevented by including both adenosine (10 mM) and PO₄ (25 mM) in the perfusate. Under these conditions kidney cortex ATP levels were more than double control values — 3.5 μmol/g wet wt. Both adenosine and PO₄ were necessary since omission of one substance resulted in no net synthesis of ATP. Furthermore, these high levels of ATP were obtained only if adequate concentrations of adenosine were maintained during perfusion. Following 3 days of perfusion the adenosine level in the perfusate decreased to about 1 mM and under this condition ATP levels were low. Adenosine levels were maintained in the perfusate by two methods: (1) addition of fresh perfusate or (2) pretreatment of the kidney with the adenosine deaminase inhibitor—deoxycoformycin. The increased levels of ATP appear directly related to the availability of nucleotide precursors and the presence of inhibitors of the enzymes involved in the catabolism of nucleotides and nucleosides (PO₄ and deoxycoformycin). Mitochondrial activity was similar in kidneys with high or low ATP levels following 5 days of preservation.     

Flexibility and length of human bronchial mucin studied using low-shear viscometry, birefringence relaxation analysis, and electron microscopy

The glycoprotein mucin was isolated from the sputum of patients with chronic obstructive bronchitis. The fractionation procedure included treatment with 6M urea at pH 12.5 followed by gel filtration in 6M urea at neutral pH. (1) Using a low-shear Cartesian diver viscometer, we found that the mucin intrinsic viscosity equals (0.32 ± 0.03) L/g in 1000 mM NaCl solution increasing to (12 ± 3) L/g in 0.1 mM NaCl (pH 7 and 20°C). (2) The relaxation of electrically induced birefringence in mucin solutions was measured and the relaxation spectrum calculated using a Fourier-transform deconvoltion method. We found that the dominant relaxation time increased from 1 to 150 μs when the exitation pulse duration used was increased from 2 to 300 μs. (3) Mucin was vacuum-dried from glycerol-containing solutions followed by low-angle rotary shadowing and electron microscopy. Mucin was found to be unbranched, with contour lengths ranging from 300 to 2500 nm and with an average of 900 nm. Our result indicate that mucin is an extended and flexible molecule with Kuhn length 0.3–0.5% of the contour length.     

Thermodynamic characterization of ethidium bromide binding to a unique site on yeast tRNAphe.

A self-consistent thermodynamic characterization of the binding of ethidium to yeast phenylalanine-specific tRNA at 25°C, pH 7.0, in 11 nM MgCl₂, 375 nM NaCl, and 25 mM sodium phosphate has been obtained. Two ethidium molecules bind per tRNA under these conditions. The stronger site has a dissociation constant equal to 1.9 ± 0.5 μM and ΔH_dis°′ = 12 ± 1 Kcal/mol, and the weaker sites has a dissociation constant equal to 24 ± 9 μM and ΔH_dis°′ = 8.9 ± 1.5 Kcal/mol. The average calorimetric ΔH_dis°′ for the to sites 10.6 ± 0.4 kcal/mol. The thermodynamics of binding to the stranger sites are most probably the thermodynamics of interaction between A·U (6) and A·U (7), the unique site identified by Jones and Kearns. The binding is enthalpically driven and classical hydrophobic interactions do not appear to be important in the binding reaction.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Energetic metabolism during acute stretch-related atrial fibrillation

Background and methods Perturbations in energetic metabolism and impaired atrial contractility may play an important role in the pathogenesis of atrial fibrillation (AF). Besides, atrial stretch is commonly associated with AF. However, the atrial energetics of stretch-related AF are poorly understood. Here, we measured indicators of energy metabolism during acute stretch-related AF. PCr, adenine nucleotides, and derivatives concentrations as well as the activity of the F₀F₁-ATPase and Na,K-ATPase were obtained after 1 h of stretch and/or AF in isolated rabbit hearts and compared to control hearts without stretch and AF. Results After 1 h of stretch-related AF, the total adenine nucleotides’ pool was significantly lower (42.2 ± 2.6 vs. 63.7 ± 8.3 μmol/g protein in control group, P < 0.05) and the PCr/ATP ratio significantly higher (2.3 ± 0.3 vs. 1.1 ± 0.1 in control group P < 0.05), because of ATP, ADP, and AMP decrease and PCr increase. The sum of high-energy phosphate compounds did not change. There were no significant differences in F₀F₁-ATPase nor Na,K-ATPase activity between the groups. Conclusions Results show that in this experimental model, acute stretch-related AF induces specific modifications of atrial myocytes energetics that may play a pivotal role in the perpetuation of the arrhythmia.     

Avocado shoot culture, plantlet development and net CO2 assimilation in an ambient and CO2 enhanced environment

Summary The proliferation and survival of avocado nodal cultures of juvenile origin were affected by the form and concentration of nitrogen. Optimum growth was achieved on modified Murashige and Skoog medium containing 67% KNO₃ and 33% NH₄NO₃ with total N of 40 mM supplemented with 100 mg l⁻¹ myo-inositol, 1 mg l⁻¹ thiamine HCl, 30 g l⁻¹ sucrose, and 4.44 μM BA with a 16-h photoperiod (120–150 μmol m⁻² s⁻¹). Proliferating shoots and plantlets were photosynthetically active. Better shoot growth and accumulation of higher biomass occurred in a CO₂-enriched environment than under ambient CO₂ conditions. CO₂ assimilation efficiency, however, was higher under the latter conditions than in a CO₂-enhanced environment, e.g., 31±7 and 17±2 μmol CO₂ m⁻² s⁻¹, respectively. The net CO₂ assimilation rates of in vitro grown plantlets were comparable to those of seedlings ex vitro.     

ATPase activity in plasmalemma-rich vesicles isolated by aqueous two-phase partitioning from Vicia faba mesophyll and epidermis: Characterization and influence of abscisic acid and fusicoccin

Plasmalemma-rich microsomal vesicles were prepared from whole leaf and acid-washed epidermal tissue of Vicia faba L. cv. Osnabrücker Markt by aqueous two-phase partitioning in dextran T-500 and polyethylenglycol 1350 aqueous phases. These vesicles were tightly sealed and predominantly right-side out, and contained a K⁺ -stimulated, mg²⁺-dependent and vanadate-sensitive ATPase. The enzyme from both tissues exhibited nearly identical properties: pH optimum 6.4, K_m for ATP 0.60 mM(whole leaf) and 0.67 mM (epidermis). V_max -480 nmol (mg protein)¹ min¹ (whole leaf) and 510 nmol (mg protein)¹ min¹ (epidermis), I₅₀ (Na₃,VO₄) 7.5 μM (whole leaf) and 15 μM (epidermis). The enzyme was not inhibited by NO₃(50 mM)or sodium azide (I mM). DCCD (20 μM) reduced enzyme activity to 50% (whole leaf) and 58% (epidermis), gramicidin S (20 μM) to 36% (whole leaf) and 41%(epidermis). Ca²⁺ inhibited the ATPase [I₅₀, C²⁺: 0.5 mM(whole leaf) and 0.8 mM(epidermis)]. Ca²⁺ inhibited the ATPase [I₅₀, C²⁺ 0.5 mM(whole leaf) und 0.8 (epidermis)]. The vanadate-sensitive ATPase from whole leaf and epidermal tissue was slightly but significantly stimulated by fusicoccin (FC) at a concentration (0.13 μM) promoting stomatal opening. The stimulation was not seen in the solubilized ATPase. Stomata of the cultivar used here were insensitive lo (±)ABA up to 2 μM level which is effective in most other cultivars and species. Likewise, at this concentration no effect of ABA on the activity of the epidermal ATPase was observed. The data are discussed with respect to the interaction of FC and ABA with the ATPase.     

SEASONAL VARIATIONS OF PHOTOSYNTHETIC PIGMENTS, TOTAL C, N, AND P CONTENT, AND PHOTOSYNTHESIS IN PHYLLARIOPSIS PURPURASCENS: (PHAEOPHYTA) FROM THE STRAIT OF GIBRALTAR

Photosynthetic pigments, C, N, and P tissue composition, and photosynthetic rate were measured from April to October in the brown alga Phyllariopsis purpurascens (C. Agardh) Henry et South (Laminariales, Phaeophyta) growing at a 30-m depth in the Strait of Gibraltar. Ir-radiance reaching the population ranged from 13.5 to 27.5 mol.m^-2.mo^-1. The available light for this species, expressed as a percentage of the irradiance above the water, was 1.8%. Dissolved inorganic nitrogen forms, NO3-and NH₄+, were constant from April to October, whereas phosphate was depleted in August. Chlorophyll a decreased from 520.0 ± 165.0 to 199.6 ± 159.9 μg.g^-1 dry weight; in contrast, chlorophyll c and carotenoids did not change until September but increased threefold in October. C:N and N:P ratios changed in the same way and in the same range. They were constant until July but increased from 15–17 up to 42 (C:N) and from 14 to 40 (N:P) in October, suggesting a severe P limitation of growth of this species. The dark respiration rate and the light compensation point were constant from April to October (0.5 ± 0.1 μmol O₂. m^-2.s^-1 and 6.5 ± 0.2 μmol.m^-2. s^-1, respectively), whereas the maximum rate of apparent photosynthesis, light onset saturation parameter, and half saturation constant for light were maximum in April to May (3.7 μmol O₂. m^-2.s^-1and 40 and 41.5 μmol.m^-2. s^-1, respectively) and October (3.6 μmol O₂. m^-2.s^-1 and 50 and 53.7 μmol.m^-2. s^-1, respectively). They were minimum in August (1.2 μmol O₂.m^-2.s^-1 and 11.3 and 12 μmol.m^-2.s^-1, respectively). These minimum figures yielded a negative carbon budget in August and 0 in September, whereas it was positive the rest of the year. Photosynthetic efficiency, estimated by the ratio between maximum apparent photosynthesis and light half saturation constant, showed a strong agreement with productivity measured by means of an independent method. These results indicate that lamina expansion in this species is controlled by photosynthetic efficiency.     

A role for membrane transport in modulation of intramuscular free glutamine turnover in streptozotocin diabetic rats

We wished to examine the effects of diabetes on muscle glutamine kinetics. Accordingly, female Wistar rats (200 g) were made diabetic by a single injection of streptozotocin (85 mg/kg) and studied 4 days later; control rats received saline. In diabetic rats, glutamine concentration of gastrocnemius muscle was 33% less than in control rats: 2.60 ± 0.06 μmol/g vs. 3.84 ± 0.13 μmol/g (P < 0.001). In gastrocnemius muscle, glutamine synthetase activity (V_max) was unaltered by diabetes (approx. 235 nmol/min per g) but glutaminase V_max increased from 146 ± 29 to 401 ± 94 nmol/min per g; substrate K_m values of neither enzyme were affected by diabetes. Net glutamine efflux (AZ concentration difference × blood flow) from hindlimbs of diabetic rats in vivo was greater than control values (?30.0 ± 3.2 vs. ?1.9 ± 2.6 nmol/min per g (P < 0.001) and hindlimb NH₃ uptake was concomitantly greater (about 27 nmol/min per g). The glutamine transport capacity (V_max) of the Na-dependent System N^m in perfused hindlimb muscle was 29% lower in diabetic rats than in controls (820 ± 50 vs. 1160 ± 80 nmol/min per g (P < 0.01)), but transporter K_m was the same in both groups (9.2 ± 0.5 nM). The difference between inward and net glutamine fluxes indicated that glutamine efflux in perfused hindlimbs was stimulated in diabetes at physiological perfusate glutamine (0.5 mM); ammonia (1 mM in perfusate) had little effect on net glutamine flux in control and diabetic muscles. In Intramuscular Na⁺ was 26% greater in diabetic (13.2 μmol/g) than control muscle, but muscle K⁺ (100 μmol/g) was similar. The accelerated rate of glutamine release from skeletal muscle and the lower muscle free glutamine concentration observed in diabetes may result from a combination of; (i), a diminished Na⁺ electrochemical gradient (i.e., the net driving force for glutamine accrual in muscle falls); (ii), a faster turnover of glutamine in muscle and (iii), an increased V_max/K_m for sarcolemmal glutamine efflux.     

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture. Serine incorporation into serine phosphoglycerides: base exchange and decarboxylation patterns.

The patterns of serine metabolism into phospholipids of cultured brain cells was examined. Labeled serine was incorporated predominantly into serine- ad ethanolamine-containing phospholipids and sphingolipids. The highest rates of labeling were observed in the (1)acyl-(2)acyl- and (1)alkyl-(2)acyl-serine phosphoglyceride fractions. Serine incorporation into both compounds appears to proceed via a base exchange mechanism. A decrease in the rate of serine phosphoglycerides labeling and a depletion of the ATP levels were observed when oligomycin or the calcium ionophore A23187 was added to the incubation medium. The inhibition of serine incorporation by A23187 could be partially reversed following addition of 10 mM CaCl2. Based on these findings it is suggested that in addition to demonstrating the energy-independent calcium-stimulated pathway, there may also be an energy related pathway. Formation of ethanolamine phosphoglycerides, as a result of serine phosphoglycerides decarboxylation, has been analyzed by using a simplified compartmental model. Of the 0.67 nmol/mg of protein turned over per h in the diacylserine phosphoglyceride compartment, 0.14 nmol/mg of protein are converted into the ethanolamine phosphoglycerides. In a similar manner, of the 0.09 nmol/mg of protein turned over per h in the (1)alkyl-(2)acyl-serine phosphoglyceride compartment, 0.014 nmol/mg of protein is converted into the (1)alkyl-(2)acyl-ethanolamine phosphoglyceride. These figures provide a first indication that a considerable portion of the ethanolamine phosphoglycerides in cultured brain cells is formed via a direct decarboxylation of the serine phosphoglycerides. In estimating the rates of (1)alkenyl-(2)acyl-ethanolamine phosphoglyceride formation from (1)alkyl-(2)acyl-ethanolamine phosphoglyceride the precursor-product specific activity crossover point could not be established. Mathematical analysis, however, enabled us to estimate the flux from the former into the latter as 0.04 nmol/mg of protein per h. A scheme for the possible metabolic interconversions of the ether bond containing serine and ethanolamine phosphoglycerides is proposed.     

The Val103Ile Polymorphism of Melanocortin‐4 Receptor Regulates Energy Expenditure and Weight Gain

Objective: The melanocortin‐4 receptor (MC4R) regulates energy intake. On the basis of animal studies, it may also regulate energy expenditure. Research Methods and Procedures: The effect of the Val103Ile polymorphism of the MC4R gene on energy metabolism was studied in 229 middle‐aged nondiabetic subjects (Group 1, age 51.2 ± 9.8 years, BMI 26.8 ± 4.5 kg/m²) and on weight gain in 1013 elderly subjects (Group 2, age 69.9 ± 2.9 years, BMI 27.4 ± 4.1 kg/m²) during a 3.5‐year follow‐up study. In Group 1, insulin sensitivity, energy expenditure, and substrate oxidation were measured with the hyperinsulinemic euglycemic clamp combined with indirect calorimetry. Results: In Group 1, the Val103Ile genotype was associated with high rates of energy expenditure (63.42 ± 13.40 in eight subjects with the Val103Ile genotype vs. 59.86 ± 7.33 J/kg per minute in 221 subjects with the Val103Val genotype, p = 0.007), high rates of glucose oxidation (8.90 ± 6.15 vs. 6.07 ± 4.38 μmol/kg per minute, p = 0.020), and low levels of free fatty acids (0.45 ± 0.18 vs. 0.56 ± 0.23 mM, p = 0.029) in the fasting state, and with high rates of glucose oxidation during the clamp (18.88 ± 4.63 vs. 17.60 ± 3.24 μmol/kg per minute, p = 0.031). In Group 2, the 103Ile allele was associated with an increase in weight gain during the follow‐up (0.78 ± 3.98 vs. ?0.82 ± 3.98 kg, p = 0.038). Discussion: The Val103Ile polymorphism of the MC4R gene is associated with energy expenditure in humans. Furthermore, it may associate with glucose oxidation, free fatty acid levels, and weight gain.     

Metabolic recovery in Atlantic salmon fry and parr following forced activity

Atlantic salmon Salmo salar fry and parr were subjected to 5 min of forced activity and the subsequent changes in oxygen consumption and ammonia excretion rates were evaluated over a 24 h period. In a second experiment, individual Atlantic salmon fry and parr were freeze‐clamped in liquid nitrogen, before, immediately following a 5 min activity period, or after periods of recovery up to 2 h. Samples were analysed for whole body phosphocreatine (PCr), ATP and lactate. Five minutes of forced activity resulted in significant increases in both oxygen consumption and ammonia excretion rates. Changes in the oxygen consumption rates were greater in the parr compared with the fry. In contrast, the post‐exercise ammonia excretion rates were nearly twice as high for the fry compared with the parr. Exercise also caused a marked decrease in PCr levels (c. 47 and 65% in fry and parr, respectively), no change in ATP levels and a significant increase in lactate levels in Atlantic salmon fry and parr. Recovery of PCr occurred quickly (between 15 and 30 min) in fry and parr. Although the post‐activity levels of lactate were lower in fry (c. 3 μmol g^?1) compared with parr (c. 14 μmol g^?1), lactate levels returned to control levels within 60 min in fry, but it took >2 h for this metabolite to recover in parr. Compared with parr, these findings show that Atlantic salmon fry possess a reduced anaerobic capacity, and these results are consistent with the theoretical and experimental evidence that smaller fish support burst swimming through aerobic processes.     

Hepatic Gluconeogenesis and the Activity of PDH in Individual Tissues of GTG-Obese Mice Following Adrenalectomy

Adrenalectomy (ADX) lowers circulating glucose levels in animal models of non-insulin dependent diabetes (NIDDM) and obesity. To investigate the role of hepatic glucose production (HGP) and tissue glucose oxidation in the improvement in glucose tolerance, hepatocyte gluconeogenesis and the activity of pyruvate dehydrogenase (PDH) were examined in different tissues of gold thioglucose (GTG) obese mice 2 weeks after ADX or sham ADX. GTG-obese mice which had undergone ADX weighed significantly less than their adrenal intact counterparts (GTG ADX: 37.5 ± 0.7g; GTG: 44.1 ± 0.4g; p<0.05), and demonstrated lower serum glucose (GTG ADX: 22.5 ± 1.6 mmol/L; GTG: 29.4 ± 1.9 mmol/L; p<0.05) and serum insulin levels (GTG ADX: 76 ± 10μ.U/mL; GTG: 470 ± 63μU/mL; p<0.05). Lactate conversion to glucose by hepatocytes isolated from ADX GTG mice was significantly reduced compared with that of hepatocytes from GTG mice (GTG ADX: 125 ± 10 nmol glucose/10⁶ cells; GTG: 403 ± 65 nmol glucose/10⁶ cells; p<0.05). ADX also significantly reduced both the glycogen (GTG ADX: 165 ± 27 μmol/liver; GTG: 614 ± 60 pmol/Iiver; p<0.05) and fatty acid content (GTG ADX: 101 ± 9 mg fatty acid/g liver; GTG: 404 ± 40 mg fatty acid/g liver; p<0.05) of the liver of GTG-obese mice. ADX of GTG-obese mice reduced PDH activity by varying degrees in all tissues, except quadriceps muscle. These observations are consistent with an ADX induced decrease in hepatic lipid stores removing fatty acid-induced increases in gluconeogenesis and increased peripheral availability of fatty acids inhibiting PDH activity via the glucose/fatty acid cycle. It is also evident that the improvement in glucose tolerance which accompanies ADX of GTG-obese mice is not due to increased PDH activity resulting in enhanced peripheral glucose oxidation. Instead, it is more likely that reduced blood glucose levels after ADX of GTG-obese mice are the result of decreased gluconeogenesis in the liver.