Changes in glucose tolerance and leptin responsiveness of rats offered a choice of lard, sucrose, and chow

Rats offered chow, lard, and 30% sucrose solution (choice) rapidly become obese. We tested metabolic disturbances in rats offered choice, chow+lard, or chow+30% sucrose solution [chow+liquid sucrose (LS)] and compared them with rats fed a composite 60% kcal fat, 7% sucrose diet [high-fat diet (HFD)], or a 10% kcal fat, 35% sucrose diet [low-fat diet (LFD)]. Choice rats had the highest energy intake, but HFD rats gained the most weight. After 23 days carcass fat was the same for choice, HFD, chow+lard, and chow+LS groups. Glucose clearance was the same for all groups during an intraperitoneal glucose tolerance test (GTT) on day 12, but fasting insulin was increased in choice, LFD fed, and chow+LS rats. By contrast, only choice and chow+LS rats were resistant to an intraperitoneal injection of 2 mg leptin/kg on day 17. In experiment 2 choice rats were insulin insensitive during an intraperitoneal GTT, but this was corrected in an oral GTT due to GLP-1 release. UCP-1 protein was increased in brown fat and inguinal white fat in choice rats, and this was associated with a significant increase in energy expenditure of choice rats during the dark period whether expenditure was expressed on a per animal or a metabolic body size basis. The increase in expenditure obviously was not great enough to prevent development of obesity. Further studies are required to determine the mechanistic basis of the rapid onset of leptin resistance in choice rats and how consumption of sucrose solution drives this process.     

The effects of a constant T3 level and thermoneutrality in diet-induced hyperphagia

Rats were thyroidectomized, then fitted with a miniosmotic pump infusing T3, thereby assuring a constant circulating level of T3. After a ten-day recovery period, they were submitted either to a chow or to a cafeteria diet. Body weight, food intake, and energy expenditure were recorded during a thirty day period. Thyroidectomized T3 supplemented rats did not exhibit hyperphagia when fed a cafeteria diet. Despite this puzzling normophagia, they still chose nutrients in a distribution similar to that of other cafeteria-fed rats and, though maintaining the same weight as chow-fed rats, increased their proportion of fatty weight compared to these rats. The relationship between energy expenditure, T3 concentration, and cafeteria diet are discussed.     

Profiling of Energy Metabolism in Olanzapine‐Induced Weight Gain in Rats and Its Prevention by the CB1‐Antagonist AVE1625

This is the first study to examine the effect of subchronic olanzapine (OLZ) on energy homeostasis in rats, covering all aspects of energy balance, including energy intake as metabolizable energy, storage, and expenditure. We further analyzed whether, and by which mechanism, the CB1‐antagonist AVE1625 might attenuate OLZ‐induced body weight gain. For this purpose, we selected juvenile female Hanover Wistar rats that robustly and reproducibly demonstrated weight gain on OLZ treatment, accepting limitations to model the aberrations on lipid and carbohydrate metabolism. Rats received 2 mg/kg OLZ orally twice daily for 12 days. Body weight and body composition were analyzed. Moreover daily food intake, energy expenditure, and substrate oxidation were determined in parallel to motility and body core temperature. OLZ treatment resulted in substantial body weight gain, in which lean and fat mass increased significantly. OLZ‐treated rats showed hyperphagia that manifested in increased carbohydrate oxidation and lowered fat oxidation (FO). Energy expenditure was increased, motility decreased, but there was no indication for hypothermia in OLZ‐treated rats. Coadministration of OLZ and AVE1625 (10 mg/kg orally once daily) attenuated body weight gain, diminishing the enhanced food intake while maintaining increased energy expenditure and decreased motility. Our data reveal that energy expenditure was enhanced in OLZ‐treated rats, an effect not critically influenced by motility. Energy uptake, however, exceeded energy expenditure and led to a positive energy balance, confirming hyperphagia as the major driving factor for OLZ‐induced weight gain. Combination of OLZ treatment with the CB1‐antagonist AVE1625 attenuated body weight gain in rats.     

Time-Dependent Effects of Progressive Gamma -Linolenate Feeding on Hyperphagia,Weight Gain,and Erythrocyte Fatty Acid Composition During Growth of Zucker Obese Rats

Obese Zucker rats (fa/fa) have low levels of arachidonic acid (AA) in liver phospholipids (PL). We have previously shown that a 70% gamma-linolenate concentrate (GLA; an AA intermediate) fed at a fixed dose (0.07 g/day) normalized hepatic PL AA and reduced weight gain selectively in the obese animals. In a follow-up study, 16 obese (fa/fa) and 16 lean (Fa/Fa) 4-week-old male rats were randomized into 4 groups of 8 each and gavaged daily with soybean oil (SOY) containing 55% 18:2ω6 (an AA precursor) or GLA, using a progressive dose (≤ 5% of total calories) based on body weight. A defined diet with 11% of energy as SOY was fed ad libitum for 60 days. GLA obese had lower body weight (p<0.0001) and 60-day cumulative food intake (p<0.05) compared to SOY obese, but neither parameter differed between the lean groups. For the last twenty days cumulative food intake was identical for GLA obese and SOY lean, whereas SOY obese consumed 18% more (p<0.05). Thus the progressive dose of GLA selectively suppressed hyperphagia in obese Zucker rats. Erythrocytes collected at 15-day intervals showed parallel increases in AA in both genotypes over time, suggesting normal AA availability during rapid growth. Thus, the reduced PL AA in the livers from the obese rats probably reflects impaired distribution in selected tissues rather than reduced hepatic production. Due to the potential health risks of enriching tissue lipids with AA, great caution is advised in considering GLA as therapy for human obesity.     

Acutely decreased thermoregulatory energy expenditure or decreased activity energy expenditure both acutely reduce food intake in mice

Despite the suggestion that reduced energy expenditure may be a key contributor to the obesity pandemic, few studies have tested whether acutely reduced energy expenditure is associated with a compensatory reduction in food intake. The homeostatic mechanisms that control food intake and energy expenditure remain controversial and are thought to act over days to weeks. We evaluated food intake in mice using two models of acutely decreased energy expenditure: 1) increasing ambient temperature to thermoneutrality in mice acclimated to standard laboratory temperature or 2) exercise cessation in mice accustomed to wheel running. Increasing ambient temperature (from 21°C to 28°C) rapidly decreased energy expenditure, demonstrating that thermoregulatory energy expenditure contributes to both light cycle (40±1%) and dark cycle energy expenditure (15±3%) at normal ambient temperature (21°C). Reducing thermoregulatory energy expenditure acutely decreased food intake primarily during the light cycle (65±7%), thus conflicting with the delayed compensation model, but did not alter spontaneous activity. Acute exercise cessation decreased energy expenditure only during the dark cycle (14±2% at 21°C; 21±4% at 28°C), while food intake was reduced during the dark cycle (0.9±0.1 g) in mice housed at 28°C, but during the light cycle (0.3±0.1 g) in mice housed at 21°C. Cumulatively, there was a strong correlation between the change in daily energy expenditure and the change in daily food intake (R² = 0.51, p<0.01). We conclude that acutely decreased energy expenditure decreases food intake suggesting that energy intake is regulated by metabolic signals that respond rapidly and accurately to reduced energy expenditure.     

Changes in Creatine and Urea Formation in Rats Fasted and Fed Low Protein Diets

Changes in the time course of the urinary excretion of creatinine, creatine and urea, and the activities of kidney transamidinase and liver urea-cycle enzymes were investigated in rats fasted and fed on a 10% casein diet and 10% casein diets supplemented with 10% glycine and/or 1.4% arginine.

The urinary total-creatinine of the fasted rats increased extremely during fasting for 7 days, while that of the animals given the 10% casein diet supplemented with glycine and arginine rose exceedingly on the 3rd day and thereafter no significant change was observed. Most of the increase of total-creatinine could be accounted for by the increase of creatine. The activity of kidney transamidinase in the fasted rats decreased in the 3rd day and thereafter kept nearly constant. The transamidinase activity of rats fed on the 10% casein diet after giving a protein-free diet for 5 days increased in the 3rd day. An inverse relation was observed between the urinary creatine and the transamidinase activity. The urinary urea increased in the rats fasted or fed on the 10% casein diets with the supplement of glycine and/or arginine. In fasting, the activities of liver urea-cycle enzymes, except arginase, had a tendency of increasing with the lapse of time. The arginase activity remained more or less constant. The reason of the extreme increase of urinary creatine during starvation was discussed.     

Chronic leptin administration promotes lipid utilization until fat mass is greatly reduced and preserves lean mass of normal female rats

Leptin is a hormone synthesized and secreted from adipose tissue. To study the physiologic effects of chronic leptin treatment, normal adult female Sprague-Dawley rats were injected subcutaneously for 35 days. Twice daily injections (250 microgram/day, b.i.d.) resulted in a significant (P<0.05) decrease in food intake that was maintained for 10 days before gradually returning to control level by day 21. Leptin decreased body weight by a maximum of 12% of the initial body weight on day 22 and remained reduced for the duration of the treatment. After 35 days of treatment, visible peritoneal adipose tissue was not detected. Body composition analysis showed that chronic injection of leptin resulted in a dramatic decrease in fat content (28+/-2 to 4+/-2 g, P<0.05; mean+/-SEM) while the lean content remained unchanged. Rats pair-fed to the leptin-treated group but treated with vehicle had the same body composition (23+/-3 g fat mass) as that measured for the ad libitum fed controls. Using indirect calorimetry we observed that leptin decreased respiratory quotient and thus increased fat oxidation. Leptin also prevented energy expenditure reduction typically associated with food restriction. Leptin treatment for 35 days decreased plasma triglyceride (0.75+/-0.07 to 0.30+/-0.03 mM, P<0.05), free fatty acid (0.56+/-0.06 to 0.32+/-0.04 mM) and insulin (3.2+/-0.5 to 1. 4+/-0.4 ng/ml, P<0.05) concentrations despite the fact that food intake was normalized by day 35. Withdrawal of leptin triggered hyperphagia indicating that leptin biology remained throughout the duration of the chronic treatment. These data suggest that leptin reduces fat mass by initially decreasing appetite and by maintaining enhanced fat utilization even when food intake has returned to that of vehicle-treated control.     

Effects of acute or chronic opioid agonists and their modulation by diurnal rhythmicity and satiety states on food intake in rats.

The effects of acute or chronic treatment with mu and k opioid agonists were investigated on food intake during light (0-6 hr) and dark (6-24 hr) phases in free fed and fasted rats. In free fed rats, morphine (MOR, 5 mg/kg, ip), a mu-agonist, induced a hyperphagic response during both light and dark phases, whereas ketocyclazocine (KCZ, 1 mg/kg, ip), a k-agonist, enhanced food intake only during the light phase. Chronic MOR (x 7 days) produced a further enhancement of hyperphagia in the light phase and attenuated the dark phase response. Chronic KCZ, however, had opposite effects, i.e. tolerance to light phase hyperphagia and an enhancement in the dark phase response. In fasted rats, neither MOR nor KCZ appreciably enhanced food intake after acute administration but chronic treatment potentiated the acute opioid effects. These results are discussed in light of the role of diurnal rhythmicity, satiety states and receptor (mu and k) specificity/interactions in the opioidergic regulation of food intake.     

Increased weight gain after ovariectomy is not a consequence of leptin resistance

The positive correlation between leptin and body fat mass has caused some investigators to speculate that leptin resistance contributes to obesity. Loss of ovarian function in human and rat is associated with increased fat mass gain and increased circulating leptin levels. To study whether ovariectomy produces leptin resistance, Sprague-Dawley female rats were ovariectomized or sham operated and injected with leptin for 35 days. Ovariectomy (OVX) produced hyperphagia and increased gain in both lean and fat mass. Daily leptin injections initially decreased food intake significantly, but feeding gradually increased to a stable level by day 16 and remained at that level for the duration of study. Body composition analysis indicated that chronic injection of leptin to OVX rats dramatically decreased (P < 0.05) fat mass [30 +/- 2 (SE) g, vehicle, to 3 +/- 1 g, leptin]. Using indirect calorimetry, we observed that OVX did not change energy expenditure or total level of fuel utilization. Leptin administration increased fat utilization and prevented reduction in calorie expenditure that is typically associated with food restriction. Leptin treatment to OVX rats decreased plasma triglyceride, free fatty acid, and insulin concentrations, whereas glucose concentration was normal. Withdrawal of leptin triggered hyperphagia, indicating that leptin biology remained throughout the duration of the chronic treatment. The same dose of leptin produced qualitatively similar data in sham-operated rats. Thus we concluded that the loss of ovarian function in rats is not associated with a change in leptin sensitivity.     

Diet-induced hyperphagia in the rat is influenced by sex and exercise

Caloric intake is increased in rats fed a diet containing greater fat or sugar than that found in laboratory chow. Because such diet-induced hyperphagia has been studied primarily in sedentary male rats, our goal here was to investigate the effects of sex and exercise on caloric intake of a diet (chow supplemented with sweet milk) chosen for its ability to stimulate hyperphagia. Rats were housed individually in cages that provided access to running wheels, and daily caloric intake of chow alone and then chow plus sweet milk was monitored during sedentary and active conditions. In sedentary rats, chow intake was greater in males compared with females. Wheel running produced similar decreases in chow intake in both sexes. Availability of the chow plus milk diet increased caloric intake compared with that observed in chow-fed rats. This diet-induced hyperphagia was significantly greater in sedentary females (35.7 +/- 3.1% increase) relative to sedentary males (9.1 +/- 2.2% increase). In addition, 35% of sedentary females consuming the chow plus milk diet developed estrous cycle disruptions. Wheel running decreased intake of the chow plus milk diet in both sexes. In active males, diet-induced hyperphagia was abolished; caloric intake was reduced to that observed during chow feeding. In active female rats, diet-induced hyperphagia was attenuated but not abolished; caloric intake of the chow plus milk diet remained greater than that observed during chow feeding. We conclude that female rats are more vulnerable than male rats to this form of diet-induced hyperphagia.     

Alterations of glucose-dependent insulinotropic polypeptide (GIP) during cold acclimation

Cold acclimation is initially associated with shivering thermogenesis in skeletal muscle followed by adaptive non-shivering thermogenesis, particularly in brown adipose tissue (BAT). In response, hyperphagia occurs to meet increased metabolic demand and thermoregulation. The present study investigates the effects of cold (4 ± 1 °C) acclimation and hyperphagia on circulating and intestinal levels of gastric inhibitory polypeptide (GIP) in rats. Pair fed animals were used as additional controls in some experiments. Cold acclimation for 42 days significantly (p<0.01) increased daily food intake. There was no corresponding change in body weight. However, body weights of pair fed cold exposed rats were significantly (p<0.01) reduced compared to controls and ad libitum fed cold exposed rats. By day 42, non-fasting plasma glucose was increased (p<0.05) by chronic cold exposure regardless of food intake. Corresponding plasma insulin concentrations were significantly (p<0.01) lower in pair fed cold exposed rats. Circulating GIP levels were elevated (p<0.05) in ad libitum fed cold acclimated rats on days 18 and 24, but returned to normal levels by the end of the study. The glycaemic response to oral glucose was improved (p<0.01) in all cold exposed rats, with significantly (p<0.05) elevated GIP responses in ad libitum fed rats and significantly (p<0.05) reduced insulin responses in pair fed rats. In keeping with this, insulin sensitivity was enhanced (p<0.05) in cold exposed rats compared to controls. By the end of the study, cold acclimated rats had significantly (p<0.01) increased BAT mass and intestinal concentrations of GIP and GLP-1 compared to controls, independent of food intake. These data indicate that changes in the secretion and actions of GIP may be involved in the metabolic adaptations to cold acclimation in rats.     

Effect of intermittent fasting followed by cold on growth, formation of reserves and energy metabolism in the golden hamster

Golden hamsters (Mesocricetus auratus) were subjected to intermittent fasting for six weeks, for three of which their environmental temperature was reduced to 4 degrees C. They were compared with a control group (fed ad libitum and kept at 22 degrees C), a group subjected six weeks only to intermittent fasting at 22 degrees C and a group fed ad libitum for six weeks, but exposed to a temperature of 4 degrees C after the third week. The effect of these various nutritional and temperature regimens on growth, on food consumption, on the formation of liver glycogen and lipid reserves and on energy metabolism was studied. Adaptation to cold raised the hamsters' food intake above the hyperphagia level observed during intermittent fasting. Cold and intermittent fasting acted synergically in raising basal oxygen consumption values and reducting the amount of body lipids. They acted antagonistically on the liver glycogen concentration; when they were used simultaneously, the effect of intermittent fasting preponderated.     

Effect of a selectiveβ 2-adrenergic agonist (Cenbuterol) on energy balance and body composition in normal and protein deficient rats

In rats fed a normal (22% protein) diet, injection of clenbuterol (1 mg/kg/d for 21 d) did not affect energy intake, energy expenditure or weight gain, but reduced energetic efficiency, and fat and energy gains and increased body protein content. Presenting a low-protein (8%) diet reduced energy intake, gain and efficiency, body protein content and the mass of the gastrocnemius muscle when compared to rats fed the control diet. Injection of the protein-deficient rats with clenbuterol (1 mg/kg/d for 21 d) caused hypophagia and reduced body weight and energy gains, energy expenditure and total body fat. However, the total body content of protein was not significantly reduced and the percentage of body protein in this protein deficient, clenbuterol-treated group was greater than that of untreated rats on both the high- and low-protein diets. The ratio of body protein to fat following clenbuterol treatment was increased by over 50% in both normal and protein-deficient rats. The results show that in protein deficient animals, clenbuterol treatment may help conserve body protein at the expense of fat, resulting in a smaller, but leaner body mass.     

Energy balance and brown adipose tissue thermogenesis during chronic endotoxemia in rats

The effects of continuously administered endotoxin on 7-day energy balance were investigated in male rats. Three groups of rats were implanted with osmotic pumps; two groups received saline-filled pumps, whereas the third received endotoxin. One of the saline groups was pair fed to match the food intake of the endotoxemic rats. After 7 days, body energy and protein and fat contents of rats were determined together with the energy content of food and feces. Endotoxin infusion not only induced fever, but it also suppressed appetite and significantly decreased body weight gain. Metabolizable energy intake was reduced by approximately 20% in infected rats. Although protein and fat gains were lowest in the endotoxin group, there appeared to be a selective loss of protein when considered as percent of body weight. Percent body fat was unaltered between the groups. Energy expenditure considered in absolute (kJ) or body weight-independent (kJ/kg0.67) terms yielded similar patterns of results; expenditure (kJ) was 10 and 20% (P less than 0.05, P less than 0.01) lower in the endotoxemic and pair-fed rats, respectively, compared with controls. Hence, compared with pair-fed rats, endotoxin-infused animals had a 10% rise in their expenditure. Brown adipose tissue thermogenesis was assessed by mitochondrial binding of guanosine 5'-diphosphate, and results showed that binding was greatest in endotoxemic rats and lowest in the pair-fed animals. The present results suggest that in this endotoxemic model appetite suppression exacerbates changes in energy balance. However, the reduction in body weight gain is also dependent on a decrease in metabolic efficiency and an increase in total energy expenditure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat production and substrate oxidation in rats fed at maintenance level and during fasting

A total of 36 Wistar rats were fed a commercial diet to a stipulated live weight of 75 g (Group A), 100 g (Group B) and 225 g (Group C). All rats were measured in energy balance experiments, in which the animals were fed near maintenance level, followed by a period of fasting with measurements of the gas exchange. The rats in Group A, B and C were fasted for 2, 3 and 4 days, respectively. The minimum heat production on the last day of fasting for all groups was proportional to metabolic body weight (kg0.75) with a regression: heat production, kJ day-1 = 321 x kg0.75 (R2 = 0.994). In rats fed near maintenance level, heat production was provided by oxidation of carbohydrates in 80-85%, oxidation of protein was 10-15%, while oxidation of fat contributed less than 10%. It is suggested that in the fasting period, the contribution to the total heat production from oxidized carbohydrate and fat depended on the size of the fat depots, a large fat depot giving rise to fat oxidation. On the last day of fasting, 24, 51 and 90% of the total heat originated from fat oxidation in Group A, B and C, respectively.     

Pharmacological actions of the peptide hormone amylin in the long-term regulation of food intake, food preference, and body weight

The ability of amylin to reduce acute food intake in rodents is well established. Longer-term administration in rats (up to 24 days) shows a concomitant reduction in body weight, suggesting energy intake plays a significant role in mediating amylin-induced weight loss. The current set of experiments further explores the long-term effects of amylin (4-11 wk) on food preference, energy expenditure, and body weight and composition. Furthermore, we describe the acute effect of amylin on locomotor activity and kaolin consumption to test for possible nonhomeostatic mechanisms that could affect food intake. Four-week subcutaneous amylin infusion of high-fat fed rats (3-300 microg.kg(-1).day(-1)) dose dependently reduced food intake and body weight gain (ED(50) for body weight gain = 16.5 microg.kg(-1).day(-1)). The effect of amylin on body weight gain was durable for up to 11 wks and was associated with a specific loss of fat mass and increased metabolic rate. The body weight of rats withdrawn from amylin (100 microg.kg(-1).day(-1)) after 4 wks of infusion returned to control levels 2 wks after treatment cessation, but did not rebound above control levels. When self-selecting calories from a low- or high-fat diet during 11 wks of infusion, amylin-treated rats (300 microg.kg(-1).day(-1)) consistently chose a larger percentage of calories from the low-fat diet vs. controls. Amylin acutely had no effect on locomotor activity or kaolin consumption at doses that decreased food intake. These results demonstrate pharmacological actions of amylin in long-term body weight regulation in part through appetitive-related mechanisms and possibly via changes in food preference and energy expenditure.     

Effects of high-fat feeding and fasting on ghrelin expression in the mouse stomach

Ghrelin is a peptide identified as an endogenous ligand for the growth hormone secretagogue receptor. Studies have shown that ghrelin stimulates growth hormone, promotes food intake and decreases energy expenditure. Furthermore, feeding status seems to influence plasma ghrelin levels, as these are increased during fasting, whereas feeding and oral glucose intake reduce plasma ghrelin. This study examined whether standardized obesity and fasting affect cellular expression of ghrelin. Specimens from the gastrointestinal tract of fed or 18-h fasted, low-fat or high-fat fed (10 weeks on diet) C57BL/6J mice were studied by immunocytochemistry (ICC) for ghrelin and in situ hybridization (ISH) for ghrelin mRNA. Ghrelin was expressed in especially the corpus but also the antrum of the stomach of all groups studied. Cells positive for ghrelin and ghrelin mRNA in the stomach were reduced in high-fat fed mice. In contrast, ghrelin expression was not affected by fasting. The reduction in ghrelin expression in the high-fat fed mice was associated with a reduction in plasma levels of ghrelin, whereas after fasting, when expression rate was not altered, there was an increase in plasma ghrelin. In conclusion, ghrelin is highly expressed in the corpus and antrum of the stomach of C57BL/6J mice. This expression is reduced in obesity, whereas fasting has no effect.     

Absence of effects of short-term fasting on plasma ghrelin and brain expression of ghrelin receptors in the tilapia, Oreochromis mossambicus

Ghrelin is an important endocrine peptide that links the gastrointestinal system and brain in the regulation of food intake and energy expenditure. In human, rat, and goldfish plasma levels of ghrelin and GH are elevated in fasted animals, suggesting that ghrelin is an orexigenic signal and a driving force behind the elevated plasma levels of GH during fasting. Ghrelin's orexigenic action is mediated by the ghrelin receptor (GHS-R1a and GHS-R1b) which is localized on neuropeptide Y (NPY) neurons in the brain. Studies were undertaken to investigate the effect of short-term fasting on plasma ghrelin and brain expression of GHS-R1a, GHS-R1b, and NPY in the tilapia. Fasting for 7 days had no effect on plasma ghrelin concentrations, whereas significant increases in plasma levels of GH were observed on day 3. Fasting significantly reduced plasma levels of IGF-I on days 3 and 7, and of glucose on days 3, 5, and 7. Brain expression of ghrelin and GHS-R1b were significantly elevated in fasted fish on day 3, but were significantly reduced on day 5. This reduction was likely due to a significant increase in the expression in the fed controls on day 5 compared to day 0. No change was detected in the expression of GHS-R1a or NPY in the brain. These results indicate that ghrelin is not acting as a hunger signal in short-term fasted tilapia and is not responsible for the elevated levels of plasma GH.     

The metabolic response to prolonged walking in fed and fasted men

Six healthy men walked 37 km (23 miles) per day over a 3-lap course for each of 4 consecutive days. Subjects were allowed breakfast and an unrestricted diet was consumed after completion of the walk, but no food was consumed during or between laps. At a later date the same subjects walked over the same course after an overnight fast and without breakfast. Completion time for each lap was 139 +/- 1 min (mean +/- SE) and exercise intensity was equivalent to 17 +/- 1% VO2max. Mean 24h energy intake was 14.5 +/- 0.8 MJ during the fed walk. Estimated daily energy expenditure was 12.0 MJ. Blood glucose concentration fell significantly on the first, third and fourth days of the fed walk, but no subject became hypoglycaemic. Glucose concentration did not fall during the fasted walk and was significantly higher pre-exercise and at the end of laps one and three when compared to the first day of the fed walk. Blood alanine concentration fell significantly after the end of the first lap of each day of the fed walk but not during the fasted walk. Blood lactate levels did not change during the course of either walk. Plasma free fatty acid, glycerol and blood 3-hydroxybutyrate concentrations were unchanged during the passage of the first lap on each day of the fed walk, but all three had increased significantly by the end of the first lap of the fasted walk.(ABSTRACT TRUNCATED AT 250 WORDS)