Hepatic glycogen synthesis in farmed European seabass (Dicentrarchus labrax L.) is dominated by indirect pathway fluxes

Hepatic glycogen synthesis fluxes from direct and indirect pathways were quantified in seabass by postmortem (2)H NMR analysis of plasma water (PW) and glycogen glucosyl (2)H enrichments from (2)H-enriched seawater. Eighteen fish (28.0 ± 1.7 cm and 218.0 ± 43.0 g) were divided into three groups of 6 and studied over 24 days with transfer to 5% (2)H-seawater after day 21. Over this period, one group was fed daily with fishmeal, a second group was fasted, and a third group was fasted for 21 days followed by 3 days refeeding. Glycogen turnover and sources were determined from the ratio of glucosyl position 5 enrichment to that of plasma water (H5/PW). Glycogen levels of fed fish were significantly higher than fasted (665.4 ± 345.2 μmol.g(-1) liver versus 77.2 ± 59.5 μmol.g(-1) liver, P<0.05) while refed fish had comparable levels to fed (584.6 ± 140.4 μmol.g(-1) liver). Glycogen enrichment of fed fish was undetectable indicating negligible turnover over 3 days. For fasted fish, H5/PW was ~50% indicating that half of the glycogen had turned over via indirect pathway flux. For refed fish, H5/PW was ~100% indicating that the indirect pathway accounted for all net glycogen synthesis. Direct pathway conversion of dietary carbohydrate to glycogen was not detected in any of the groups.     

Quantitation of gluconeogenesis by (2)H nuclear magnetic resonance analysis of plasma glucose following ingestion of (2)H(2)O

We present a simple (2)H NMR assay of the fractional contribution of gluconeogenesis to hepatic glucose output following ingestion of (2)H(2)O. The assay is based on the measurement of relative deuterium enrichment in hydrogens 2 and 3 of plasma glucose. Plasma glucose was enzymatically converted to gluconate, which displays fully resolved deuterium 2 and 3 resonances in its (2)H NMR spectrum at 14.1 T. The signal intensity of deuterium 3 relative to deuterium 2 in the gluconate derivative as quantitated by (2)H NMR was shown to provide a precise and accurate measurement of glucose enrichment in hydrogen 3 relative to hydrogen 2. This measurement was used to estimate the fractional contribution of gluconeogenesis to hepatic glucose output for two groups of rats; one group was fasted for 7 h and the other was fasted for 29 h. Rats were administered (2)H(2)O to enrich total body water to 5% over the last 4-5 h of each fasting period. For the 7-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.32 +/- 0.09 (n = 7). This indicates that gluconeogenesis contributed 32 +/- 9% of total hepatic glucose output with glycogenolysis contributing the remainder. For the 29-h fasted group, the hydrogen 3/hydrogen 2 enrichment ratio of plasma glucose was 0.81 +/- 0.10 (n = 6), indicating that gluconeogenesis supplied the bulk of hepatic glucose output (81 +/- 10%).     

Resolving the sources of plasma glucose excursions following a glucose tolerance test in the rat with deuterated water and [U-13C]glucose

Sources of plasma glucose excursions (PGE) following a glucose tolerance test enriched with [U-(13)C]glucose and deuterated water were directly resolved by (13)C and (2)H Nuclear Magnetic Resonance spectroscopy analysis of plasma glucose and water enrichments in rat. Plasma water (2)H-enrichment attained isotopic steady-state within 2-4 minutes following the load. The fraction of PGE derived from endogenous sources was determined from the ratio of plasma glucose position 2 and plasma water (2)H-enrichments. The fractional gluconeogenic contributions to PGE were obtained from plasma glucose positions 2 and 5 (2)H-positional enrichment ratios and load contributions were estimated from plasma [U-(13)C]glucose enrichments. At 15 minutes, the load contributed 26±5% of PGE while 14±2% originated from gluconeogenesis in healthy control rats. Between 15 and 120 minutes, the load contribution fell whereas the gluconeogenic contribution remained constant. High-fat fed animals had significant higher 120-minute blood glucose (173±6 mg/dL vs. 139±10 mg/dL, p<0.05) and gluconeogenic contributions to PGE (59±5 mg/dL vs. 38±3 mg/dL, p<0.01) relative to standard chow-fed controls. In summary, the endogenous and load components of PGE can be resolved during a glucose tolerance test and these measurements revealed that plasma glucose synthesis via gluconeogenesis remained active during the period immediately following a glucose load. In rats that were placed on high-fat diet, the development of glucose intolerance was associated with a significantly higher gluconeogenic contribution to plasma glucose levels after the load.     

Measurement of gluconeogenesis by deuterated water: the effect of equilibration time and fasting period

Fasting gluconeogenesis (GNG) is often quantified using the 2H2O technique, which is based on plasma 2H2O enrichment and ensuing enrichment of plasma glucose at the C5 and C2 positions. Fractional (fr)GNG can be calculated using the ratio of C5 to C2 enrichment or the ratio of C5 to plasma 2H2O enrichment. For the latter, equilibration of 2H2O and C2 is required. The optimal equilibration period of 2H2O and C2 remains to be elucidated. In six healthy male subjects fasted for 18 h, we studied the effects of 3-, 5-, and 15-h 2H2O incubation periods on 1) the equilibration of plasma 2H2O and C2 glucose enrichment, 2) the measurement of frGNG, and 3) C5 labeling of hepatic glycogen after 1 mg of glucagon administration. After 3-h 2H2O incubation, plasma 2H2O and C2 were not equilibrated, frGNG C5/2H2O and C5/C2 were also different as was gluconeogenesis calculated with C5/2H2O and C5/C2. After 5- and 15-h 2H2O incubation, plasma 2H2O and C2 were equilibrated, and frGNG C5/2H2O and C5/C2 were similar, as was GNG calculated with C5/2H2O and C5/C2. After glucagon administration, no difference of C5 enrichment was found between 3, 5, and 15 h of 2H2O incubation. In conclusion, for reliable measurement of GNG in healthy subjects with C5/2H2O incubation periods longer than 3 h are required. After 5- and 15-h 2H2O incubation, GNG can be reliably measured with C5/2H2O. Gluconeogenetic labeling of glycogen did not affect the results after 3, 5, or 15 h of 2H2O incubation.     

Sources of glucose production in cirrhosis by 2H2O ingestion and 2H NMR analysis of plasma glucose

Plasma glucose 2H enrichment was quantified by 2H NMR in patients with cirrhosis (n=6) and healthy subjects (n=5) fasted for 16 h and given 2H(2)O to approximately 0.5% body water. The percent contribution of glycogenolysis and gluconeogenesis to glucose production (GP) was estimated from the relative enrichments of hydrogen 5 and hydrogen 2 of plasma glucose. Fasting plasma glucose levels were normal in both groups (87+/-7 and 87+/-24 mg/dl for healthy and cirrhotic subjects, respectively). The percent contribution of glycogen to GP was smaller in cirrhotics than controls (22+/-7% versus 46+/-4%, P<0.001), while the contribution from gluconeogenesis was larger (78+/-7% versus 54+/-4%, P<0.001). In all subjects, glucose 6R and 6S hydrogens had similar enrichments, consistent with extensive exchange of 2H between body water and the hydrogens of gluconeogenic oxaloacetate (OAA). The difference in 2H-enrichment between hydrogen 5 and hydrogen 6S was significantly larger in cirrhotics, suggesting that the fractional contribution of glycerol to the glyceraldehyde-3-phosphate (G3P)-moiety of plasma glucose was higher compared to controls (19+/-6% versus 7+/-6%, P<0.01). In all subjects, hydrogens 4 and 5 of glucose had identical enrichments while hydrogen 3 enrichments were systematically lower. This reflects incomplete exchange between the hydrogen of water and that of 1-R-dihydroxyacetone phosphate (DHAP) or incomplete exchange of DHAP and G3P pools via triose phosphate isomerase.     

Sources of blood glycerol during fasting

To determine the source(s) of blood and very low density lipoprotein (VLDL)-triglyceride glycerol during fasting, four men ingested (2)H(2)O from 14 to 20 h into a 60-h fast to achieve ~0.5% body water enrichment. At 60 h of fasting, glycerol flux was measured using [2-(14)C]glycerol. Blood was taken for measurement of (2)H enrichment at carbon 6 of glucose and at carbon 3 of free glycerol and VLDL-triglyceride glycerol. (2)H enrichment of the 2 hydrogens bound to carbon 3 of VLDL-triglyceride glycerol was 105 +/- 2% of the (2)H enrichment of the 2 hydrogens bound to carbon 6 of glucose, indicating isotopic equilibrium between hepatic glyceraldehyde 3-P and glycerol 3-P. The (2)H enrichment of the 2 hydrogens bound to carbon 3 of free glycerol was 17 +/- 3% of VLDL-triglyceride glycerol, indicating that a significant percentage of free glycerol in blood originated from the hydrolysis of circulating VLDL-triglyceride or a pool of glycerol with similar (2)H enrichment. Glycerol flux was 6.3 +/- 1.1 micromol. kg(-1). min(-1). Glycerol appearing from nonadipose tissue sources was then approximately 1.1 micromol. kg(-1). min(-1). Seven other subjects were fasted for 12, 42, and 60 h. A small percentage of glycerol in the circulation after 12 h of fasting was enriched with (2)H. The enrichment of the 2 hydrogens bound to carbon 3 of free glycerol in the longer periods of fasting was approximately 16% of the enrichment of the 2 hydrogens bound to carbon 6 of glucose. Therefore, as much as 15-20% of systemic glycerol turnover during fasting is not from lipolysis of adipose tissue triglyceride.     

Fuel metabolism in fasted newborn rabbits

Newborn rabbits delivered by Caesarean section at term were fasted for 72 h at 36 degrees C. Despite the abrupt interruption of maternal supply of energy substrates, glycaemia remains stable for 4 h after birth. This can be related to glucose production via rapid liver glycogenolysis; however, indirect evidence suggests that gluconeogenesis could also contribute to glucose production during this period. There is a selective decrease in the concentrations of gluconeogenic substrates and a suitable hormonal environment for gluconeogenesis as decreased insulin and increased glucagon concentration just after birth. The relative hypoglycaemia which develops after 6 h of life (2.6 mM at 72 h), despite high blood concentrations of non-esterified fatty acids and ketone bodies is not due to a deficient gluconeogenesis per se, as injection of gluconeogenic substrates to 72 h fasted newborns produces a three-fold increase in plasma glucose concentration. It is suggested that this relative hypoglycaemia is secondary to limited gluconeogenic substrate availability in the form of low circulting concentrations of gluconeogenic amino acids.     

Effect of fasting on nychthemeral concentrations of plasma growth hormone (GH), insulin-like growth factor I (IGF-I), and cortisol in channel catfish (Ictalurus punctatus)

This experiment was conducted to characterize the effect of fasting versus satiety feeding on plasma concentrations of GH, IGF-I, and cortisol over a nychthemeron. Channel catfish fingerlings were acclimated for two weeks under a 12L:12D photoperiod, then fed or fasted for 21 d. On day 21, blood samples were collected every 2 h for 24 h. Weight of fed fish increased an average of 66.2% and fasted fish lost 21.7% of body weight on average. Average nychthemeral concentrations of plasma GH were not significantly different between fed (24.7 ng/mL) and fasted (26.8 ng/mL) fish, but average nychthemeral IGF-I concentrations were higher in fed (23.4 ng/mL) versus fasted (17.8 ng/mL) fish. An increase in plasma IGF-I concentrations was observed in fasted fish 2 h after a peak in plasma GH, but not in fed fish. Average nychthemeral plasma cortisol concentrations were higher in fed (14.5 ng/mL) versus fasted (11.0 ng/mL) fish after 21 d. Significant fluctuations and a postprandial increase in plasma cortisol were observed in fed fish and there was an overall increase in plasma cortisol of both fasted and fed fish during the scotophase. The present experiment indicates little or no effect of 21-d fasting on plasma GH levels but demonstrates fasting-induced suppression of plasma IGF-I and cortisol levels in channel catfish.     

Metabolic responses to exercise after fasting

Fasting before exercise increases fat utilization and lowers the rate of muscle glycogen depletion. Since a 24-h fast also depletes liver glycogen, we were interested in blood glucose homeostasis during exercise after fasting. An experiment was conducted with human subjects to determine the effect of fasting on blood metabolite concentrations during exercise. Nine male subjects ran (70% maximum O2 consumption) two counterbalanced trials, once fed and once after a 23-h fast. Plasma glucose was elevated by exercise in the fasted trial but there was no difference between fed and fasted during exercise. Lactate was significantly higher (P less than 0.05) in fasted than fed throughout the exercise bout. Fat mobilization and utilization appeared to be greater in the fasted trial as evidenced by higher plasma concentrations of free fatty acids, glycerol, and beta-hydroxybutyrate as well as lower respiratory exchange ratio in the fasted trial during the first 30 min of exercise. These results demonstrate that in humans blood glucose concentration is maintained at normal levels during exercise after fasting despite the depletion of liver glycogen. Homeostasis is probably maintained as a result of increased gluconeogenesis and decreased utilization of glucose in the muscle as a result of lowered pyruvate dehydrogenase activity.     

Glucose phosphorylation is not rate limiting for accumulation of glycogen from glucose in perfused livers from fasted rats

Incorporation of Glc and Fru into glycogen was measured in perfused livers from 24-h fasted rats using [6-3H]Glc and [U-14C]Fru. For the initial 20 min, livers were perfused with low Glc (2 mM) to deplete hepatic glycogen and were perfused for the following 30 min with various combinations of Glc and Fru. With constant Fru (2 mM), increasing perfusate Glc increased the relative contribution of Glc carbons to glycogen (7.2 +/- 0.4, 34.9 +/- 2.8, and 59.1 +/- 2.7% at 2, 10, and 20 mM Glc, respectively; n = 5 for each). During perfusion with substrate levels seen during refeeding (10 mM Glc, 1.8 mumol/g/min gluconeogenic flux from 2 mM Fru), Fru provided 54.7 +/- 2.7% of the carbons for glycogen, while Glc provided only 34.9 +/- 2.8%, consistent with in vivo estimations. However, the estimated rate of Glc phosphorylation was at least 1.10 +/- 0.11 mumol/g/min, which exceeded by at least 4-fold the glycogen accumulation rate (0.28 +/- 0.04 mumol of glucose/g/min). The total rate of glucose 6-phosphate supply via Glc phosphorylation and gluconeogenesis (2.9 mumol/g/min) exceeded reported in vivo rates of glycogen accumulation during refeeding. Thus, in perfused livers of 24-h fasted rats there is an apparent redundancy in glucose 6-phosphate supply. These results suggest that the rate-limiting step for hepatic glycogen accumulation during refeeding is located between glucose 6-phosphate and glycogen, rather than at the step of Glc phosphorylation or in the gluconeogenic pathway.     

Daily changes in parameters of energy metabolism in brain of rainbow trout: dependence on feeding

We assessed the daily patterns of parameters involved in energy metabolism in plasma and brain of rainbow trout. Where daily rhythms were found, we analyzed the potential influence of feeding. Immature rainbow trout were randomly distributed in 3 groups: fish fed for 7 days, fish fasted for 7 days, and fish fasted for 7 days and refed for 4 days. On sampling day, fish of fed and refed groups were fed at 11.00 h, and all fish were sampled from each treatment group using the following time schedule: 14.00, 18.00, 21.00, 00.00, 04.00, 07.00, 10.00 and 14.00 h. The results obtained from metabolic parameters assessed in plasma and brain can be grouped into three different categories, such as (i) those displaying no 24 h changes in fed fish such as plasma lactate, protein or acetoacetate levels, as well as brain amino acid and protein levels, and lowKm(glucose) hexokinase, and aspartate aminotransferase activities, (ii) those displaying 24 h changes that were apparently dependent on feeding since they disappeared in fasted fish such as the case of plasma cortisol, glucose and triglyceride levels, as well as brain glycogen, glucose, and lactate levels, and pyruvate kinase and hexokinase IV activities, and (iii) those parameters displaying 24 h changes apparently not dependent on feeding such as plasma amino acids, brain acetoacetate levels as well as several enzyme activities measured in brain such as glucose 6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, glutamate dehydrogenase, and lactate dehydrogenase-oxidase. In general, 24 h changes dependent on feeding indicate an increased use of glucose in brain several hours post-feeding whereas those changes not dependent on feeding were characterized by reduced levels/activity at the night period suggesting a metabolic depression in brain during darkness.     

Substrate usage during prolonged exercise following a preexercise meal

The effect of a high-carbohydrate meal 4 h before 105 min of exercise at 70% of maximal O2 uptake was determined in seven endurance-trained cyclists and compared with exercise following a 16-h fast. The preexercise meal produced a transient elevation of plasma insulin and blood glucose, which returned to fasting basal levels prior to the initiation of exercise. The meal also resulted in a 42% elevation (P less than 0.05) of glycogen within the vastus lateralis at the beginning of exercise. The 1st h of exercise when subjects were fed was characterized by a 13-25% decline (P less than 0.05) in blood glucose concentration, a suppression of the normal increase in plasma free fatty acids and blood glycerol, and a 45% (P less than 0.05) greater rate of carbohydrate oxidation compared with exercise when subjects were fasted. After 105 min of exercise, there were no significant differences when subjects were fed or fasted regarding blood glucose levels, rate of carbohydrate oxidation, or muscle glycogen concentration. The greater muscle glycogen utilization (97 +/- 18 vs. 64 +/- 8 mmol glucosyl units X kg-1; P less than 0.05) and carbohydrate oxidation when subjects were fed appeared to be derived from the glycogen synthesized following the meal. These results indicate that preexercise feedings alter substrate availability despite a return of plasma insulin to fasting levels prior to exercise and that these effects persist until the 2nd h of exercise.     

Dietary methionine effects on plasma homocysteine and HDL metabolism in mice

The effects of dietary manipulation of folate and methionine on plasma homocysteine (Hcy) and high-density lipoprotein cholesterol (HDL-C) levels in wild-type and apolipoprotein-E-deficient mice were determined. A low-folate diet with or without folate and/or methionine supplementation in drinking water was administered for 7 weeks. Fasted Hcy rose to 23 microM on a low-folate/high-methionine diet, but high folate ameliorated the effect of high methionine on fasted plasma Hcy to approximately 10 microM. Determination of nonfasted plasma Hcy levels at 6-h intervals revealed a large diurnal variation in Hcy consistent with a nocturnal lifestyle. The daily average of nonfasted Hcy levels was higher than fasted values for high-methionine diets but lower than fasted values for low-methionine diets. An acute methionine load by gavage of fasted mice increased plasma Hcy 2.5 h later, but mice that had been on high-methionine diets had a lower fold induction. Mice fed high-methionine diets weighed less than mice fed low-methionine diets. Based on these results, two solid-food diets were developed: one containing 2% added methionine and the other containing 2% added glycine. The methionine diet led to fasted plasma Hcy levels of >60 microM, higher than those with methionine supplementation in drinking water. Mice on methionine diets had >20% decreased body weights and decreased HDL-C levels. An HDL turnover study demonstrated that the HDL-C production rate was significantly reduced in mice fed the methionine diet.     

IGF-I and branchial IGF receptor expression and localization during salinity acclimation in striped bass

The initial response of the IGF-I system and the expression and cellular localization of IGF type-I receptor (IGF-IR) were studied in the gill of a euryhaline teleost during salinity acclimation. Exposure of striped bass (Morone saxatilis) to hyperosmotic and hypoosmotic challenges induced small, transitory (<24 h) deflections in hydromineral balance. Transfer from freshwater (FW) to seawater (SW) induced an initial decrease in plasma IGF-I levels after 24 h in both fed and fasted fish. There was an overall decrease in liver IGF-I mRNA levels after SW transfer, suggesting that decreased plasma levels may be due to a decline in hepatic IGF-I synthesis. No changes were observed in gill IGF-I mRNA, but SW transfer induced an increase in gill IGF-IR mRNA after 24 h. Transfer from SW to FW induced an increase in plasma IGF-I levels in fasted fish. In fed fish, no significant changes were observed in either plasma IGF-I, liver, or gill IGF-I mRNA, or gill IGF-IR mRNA levels. In a separate experiment, FW-acclimated fish were injected with saline or IGF-I prior to a 24-h SW challenge. Rapid regain of osmotic balance following SW transfer was hindered by IGF-I. Immunohistochemistry revealed for the first time in teleosts that IGF-IR and Na(+)-K(+)-ATPase are localized in putative chloride cells at the base of the lamellae, identifying these cells in the gill as a target for IGF-I and IGF-II. Overall the data suggest a hyperosmoregulatory role of IGF-I in this species.     

Quantitative measurement of gluconeogenesis from isobutyrate in sheep.

Experiments with continuous infusion of [14C] isobutyrate and single injection of [3H] glucose were performed in two sheep under fed and fasted conditions in order to investigate the contribution of isobutyrate to glucose synthesis. The pool size, total entry and irreversible loss of glucose in the fed sheep were 2.8 mmol/kg0.75, 1.70 and 1.43 mmol/h per kg0.75. After 72-h fasting these parameters decreased about 40% but recycling of glucose carbon increased from 16 to 38% of the total entry rate. Isobutyrate infused intravenously at a rate of 3.5 mmol/h contributed to a minimum of 3-5% of glucose entry indicating that at least 40-60% of the infused isobutyrate was used for net glucose synthesis. The efficiency of the glucogenic and energetic use of isobutyrate as compared to propionate is discussed.     

Nutritional Status as the Key Modulator of Antioxidant Responses Induced by High Environmental Ammonia and Salinity Stress in European Sea Bass (Dicentrarchus labrax)

Salinity fluctuation is one of the main factors affecting the overall fitness of marine fish. In addition, water borne ammonia may occur simultaneously with salinity stress. Additionally, under such stressful circumstances, fish may encounter food deprivation. The physiological and ion-osmo regulatory adaptive capacities to cope with all these stressors alone or in combination are extensively addressed in fish. To date, studies revealing the modulation of antioxidant potential as compensatory response to multiple stressors are rather lacking. Therefore, the present work evaluated the individual and combined effects of salinity challenge, ammonia toxicity and nutritional status on oxidative stress and antioxidant status in a marine teleost, European sea bass (Dicentrarchus labrax). Fish were acclimated to normal seawater (32 ppt), to brackish water (20 ppt and 10 ppt) and to hypo-saline water (2.5 ppt). Following acclimation to different salinities for two weeks, fish were exposed to high environmental ammonia (HEA, 20 mg/L representing 50% of 96h LC₅₀ value for ammonia) for 12 h, 48 h, 84 h and 180 h, and were either fed (2% body weight) or fasted (unfed for 7 days prior to HEA exposure). Results show that in response to decreasing salinities, oxidative stress indices such as xanthine oxidase activity, levels of hydrogen peroxide (H₂O₂) and lipid peroxidation (malondialdehyde, MDA) increased in the hepatic tissue of fasted fish but remained unaffected in fed fish. HEA exposure at normal salinity (32 ppt) and at reduced salinities (20 ppt and 10 ppt) increased ammonia accumulation significantly (84 h–180 h) in both feeding regimes which was associated with an increment of H₂O₂ and MDA contents. Unlike in fasted fish, H₂O₂ and MDA levels in fed fish were restored to control levels (84 h–180 h); with a concomitant increase in superoxide dismutase (SOD), catalase (CAT), components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase), ascorbate peroxidase (APX) activity and reduced ascorbate (ASC) content. On the contrary, fasted fish could not activate many of these protective systems and rely mainly on CAT and ASC dependent pathways as antioxidative sentinels. The present findings exemplify that in fed fish single factors and a combination of HEA exposure and reduced seawater salinities (upto 10 ppt) were insufficient to cause oxidative damage due to the highly competent antioxidant system compared to fasted fish. However, the impact of HEA exposure at a hypo-saline environment (2.5 ppt) also defied antioxidant defence system in fed fish, suggesting this combined factor is beyond the tolerance range for both feeding groups. Overall, our results indicate that the oxidative stress mediated by the experimental conditions were exacerbated during starvation, and also suggest that feed deprivation particularly at reduced seawater salinities can instigate fish more susceptible to ammonia toxicity.     

Postprandial intestinal blood flow, metabolic rates, and exercise in Chinook salmon (Oncorhynchus tshawytscha)

Following a relatively large meal (2% body mass of dry pellets), intestinal blood flow in chinook salmon (Oncorhynchus tshawytscha) increased significantly, up to 81%, between 14 and 29 h postprandially. Also, 15 h postprandially, oxygen consumption (M(2)) was elevated by 128% compared with a measurement of routine M(2) made after 1 wk of fasting. The postprandial increase in MO(2) (the heat increment) was 33 micromol O(2) min(-1) kg(-1). Because intestinal blood flow is known to decrease during swimming activity in fish, we therefore tested the hypothesis that swimming fish would have to make a trade-off between maximum swimming activity and digestive activity by comparing the swimming performance and metabolic rates of fed and fasted chinook salmon. As expected, MO(2) increased exponentially with swimming velocity in both fed and fasted fish. Moreover, the heat increment was irreducible during swimming, such that MO(2) remained approximately 39 micromol O(2) min(-1) kg(-1) higher in fed fish than in fasted fish at all comparable swimming speeds. However, maximum M dot o2 was unaffected by feeding and was identical in both fed and fasted fish (approximately 250 micromol O(2) min(-1) kg(-1)), and, as a result, the critical swimming speed (U(crit)) was 9% lower in the fed fish. Three days after the fish were fed and digestion was completed, MO(2) and U(crit) were not significantly different from those measured in fasted fish. The ability of salmonids to maintain feeding metabolism during prolonged swimming performance is discussed, and it is suggested that reduced swimming performance may be due to postprandial sparing of intestinal blood to support digestion, thereby limiting the allocation of blood flow to locomotory muscles.     

Metabolic responses to acute handling by fingerling inland and anadromous striped bass

Fed and 3-day fasted inland (average mass: 6.97 g) and anadromous (average mass: 6.54 g) striped bass Morone saxatilis fingerlings were held in dipnets above water for 5 min in groups of six. Severity of the response to this handling was measured by whole-body glucose, glycogen, and lactic acid in non-handled bass (considered control level), and then at 30 min, 1, 6, 12, 24 and 48 h recovery. At resting levels, both fed and fasted inland bass showed significantly higher concentrations of the whole body variables than anadromous bass. All four groups of bass showed an increase in lactic acid and glucose immediately after handling, with a concomitant decrease in glycogen. Peak levels of glucose and lactic acid were similar in the four groups. Fasting did not have an effect on the glucose and lactic acid responses, but did affect the glycogen response. The two fasted groups did not return to control glycogen concentrations during the 48-h recovery period. By 48 h, both glucose and lactic acid had returned to control levels. It is concluded that inland and anadromous strains of fingerling striped bass do not differ in their sensitivity to an acute handling stress. Recovery of glycogen energy stores following handling is much better if fish are not fasted before handling.     

Simplified analysis of acetaminophen glucuronide for quantifying gluconeogenesis and glycogenolysis using deuterated water

Measurement of acetaminophen glucuronide (AG) ²H enrichment from deuterated water (²H₂O) by ²H nuclear magnetic resonance (NMR) analysis of its monoacetone glucose (MAG) derivative provides estimation of gluconeogenic and glycogenolytic contributions to endogenous glucose production (EGP). However, AG derivatization to MAG is laborious and unsuitable for high-throughput studies. An alternative derivative, 5-O-acetyl monoacetone glucuronolactone (MAGLA), was tested. Eleven healthy subjects ingested ²H₂O to 0.5% body water enrichment and 500 mg of acetaminophen. Plasma glucose and urinary glucuronide positional ²H enrichments were measured by ²H NMR spectroscopy of MAG and MAGLA, respectively. A Bland–Altman analysis indicated agreement at the 95% confidence level between glucose and glucuronide estimates.     

Influence of fasting on glycogen depletion in rats during exercise

We recently observed that a 24-h fasted group of rats could run longer than an ad libitum fed control group before becoming exhausted. Because of the demonstrated importance of glycogen levels and free fatty acid availability during endurance exercise, we have investigated several parameters of carbohydrate and lipid metabolism in exercised and nonexercised rats that were either fed ad libitum or fasted for 24 h. A 24-h fast depleted liver glycogen, lowered plasma glucose concentration, decreased muscle glycogen levels, and increased free fatty acid and beta-hydroxybutyrate concentrations in plasma. During exercise the fasted group had lower plasma glucose concentration, higher plasma concentration of free fatty acids and beta-hydroxybutyrate, and a lower muscle glycogen depletion rate than did the ad libitum fed group. Since fasted rats were able to continue running even when plasma glucose had dropped to levels lower than those of fed-exhausted rats, it seems unlikely that blood glucose level, per se, is a factor in causing exhaustion. These results suggest that fasting increases fatty acid utilization during exercise and the resulting "glycogen sparing" effect may result in increased endurance.