Gastrointestinal and metabolic consequences of a rat's meal on maintenance diet ad libitum

Physiology & Behavior, Vol. 27, pp. 929-939. PergamonPress and BrainResearch Publ., 1981. Printedin the U.S.A.

Gastrointestinal and Metabolic C o n s e q u e n c e s of a Rat's Meal on Maintenance Diet Ad Libitum 1 J. C. N E W M A N 2 A N D D.A. B O O T H 3 Biochemistry Laboratory, D e p a r t m e n t o f Psychology University o f Birmingham, P.O. Box 363, Birmingham B I 5 2TT, England R e c e i v e d 19 O c t o b e r 1979 NEWMAN, J. C. AND D. A. BOOTH. Gastrointestinal and metabolic consequences of a rat's meal on maintenance diet ad libitum. PHYSIOL. BEHAV. 27(5) 929-939, 1981.--The gastric emptying of normally sized chow meals taken after minimal food deprivation appeared to be almost constant in rate for most of the period of emptying, although a short initial acceleration was not excluded and a slowing was generally observed late in emptying. Intestinal contents of dry matter and carbohydrate remained about constant and so under these conditions absorption rate equalled the recent gastric emptying rate. When the rat's meals are frequent, in the dark period of the 24 hour, the stomach emptied much more rapidly. When measured post mortem in groups of rats, blood concentrations of glucose did not vary markedly after a meal in the dark period, nor did blood concentrations of alanine, glycerol and other gluconeogenic precursors. An increase in rates of cerebral glucose uptake and conversion to glutamate and lactate was occasionally observed after meals, by comparisons of specific activities 5 min after subcutaneous injection of U-14C-glucose, but the increase was not regionally localised. Hepatic glycogen concentration did not vary up to the time the next meal would have been taken in the mid dark period. However, gluconeogenic capacity was reduced by 20--30%for about 90 min following the meal, as measured by conversion of a loading dose of 14C-alanineto ~4C-glucose in blood. Gluconeogenesis or regulation of hepatic glucose output may protect the brain and other tissues, including even the liver, from the minor and brief variations in absorption between meals ad lib. Normal satiety and hunger may be anticipatory responses, established by the metabolic and/or hormonal consequences of occasional bursts or drops in absorption rate. Gastric emptying pattern Absorption rate Blood metabolites Gluconeogenesis and feeding Satiety signals

G A S T R O I N T E S T I N A L processing is clearly a major influence on the rates of substrate supply to tissues and on the secretion of hormones from the gut and the pancreas that in turn act on gastrointestinal processes and the distribution of absorbed substrates [7]. Variations and non-linearities in gastric emptying and its effects on absorption, hormones and neural signals could have major physiological and behavioural consequences. Rapid phases of absorption would increase the deposition of energy relative to its utilization, thus promoting weight gain and obesity [8, 9, 10]. Transiently rapid absorption shortly after a meal, and a slowing of absorption later as the stomach empties, could be the ultimate sources of a number of physiological controls of the meal pattern, for many learned and unlearned satiation and hunger signals relate to intermediary metabolism [1, 4, 5, 8, 10, 17, 25, 55]. For example, one of the strongest satiety signals seems to depend on the rapid oxidation of absorbed

Brain glucose uptake

carbohydrate, acting directly [9] and indirectly via conditioning [6]. In the rat maintained on standard laboratory diet, the major energy-yielding nutrient available for both fat disposition and satiety/hunger is glucose from the digestion of starch. Yet the pattern in which the stomach empties after a meal on maintenance chow ad lib has not hitherto been systematically assessed in the laboratory rat. Gastric emptying has been measured after unphysiological liquid loads, prolonged fasting, or abnormally large meals: as in man, the emptying pattern under such conditions has a marked negative acceleration and has been fitted to exponential, square root or reciprocal cube root functions [10, 22, 40, 52]. An initially very rapid delivery of food to the intestine is seen in fasted rats, resulting in substantial temporary accumulation of intestinal contents [57] and therefore presumably a subsequent rapid phase of absorption. Such "dumping" might not

1Supported by Project Grant G974/051/B from the U.K. Medical Research'Council to D.A. Booth. With the technical assistance of Veronica Nolan. 2Present address: Thanet Technical College, Ramsgate, Kent, England. aTo whom reprint requests should be forwarded.

C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s I n c . - - 0 0 3 1 - 9 3 8 4 / 8 1 / I 10929-11502.00/0

930

occur in freely fed rats, because of the inhibitory feedback from the intestinal wall which is present during the digestive period [22]. It seemed possible that meals ad lib sustained a relatively constant rate of absorption, unless gastric emptying rate either or both varied around the clock [91 or/and was at times fast enough to empty the stomach before its refilling by the next meal. It was therefore considered important to determine the shape of the gastric emptying curve after meals ad lib on a standard chow and also the variation in the emptying pattern around the clock. As neither quantitative radiography nor cannulation-withdrawal techniques are feasible with laboratory chow in a small animal, groups of rats at different times following meals were sampled post mortem. Certain relevant metabolic consequences of variation in absorption rate were measured in related experiments. Such measurements of tissue substrate concentrations and fluxes also require post mortem analysis and blood substrate levels were also measured in this same manner for the present studies. Hepatic intermediary metabolism is central to the utilization of carbohydrate, amino acid and fatty acid substrates [4, 5, 9, 10, 17]. Russek [42-44] proposed a mechanism by which hepatocyte glucose input or output could control the discharge rate of local glucose-sensitive nerve fibres [38]. Hepatic glycogenolysis, hepatic and renal gluconeogenesis and intestinal glucose absorption are all capable of adding glucose to this exchange (and to glucose in the circulation to the brain). Indeed, hepatic glycogen levels have been invoked in hypotheses of feeding control [43,56]. So, we assessed the variation in these three sources of glucose in the period between meals ad lib. In the rat, hepatic glycogen levels show a two-fold circadian rhythm partly dependent on prior feeding [49]. They drop sharply whenever food is withheld for several hours after a meal is due [2], at least during daytime when there is no underlying rise in glycogen content of the liver. Gluconeogenesis greatly increases during prolonged fasting (24--28 hr) [16], but is diminished again within 2 hr of glucose loading 135]. The timing of the increase in gluconeogenesis following the absorptive phase has not been closely studied but an increase was observed in vitro from 2-3 hr after the food had been removed at the start of the daylight period [46]. Furthermore, hepatic gluconeogenesis is stimulated by an increase in availability of the main precursors for glucose formation (glycerol, amino acids, lactate). The plasma concentration of glycerol increases during starvation and amino acid transport into the liver cell is increased by hormones, resulting in increased substrate availability for gluconeogenesis. Thus, as well as measuring the concentration of gluconeogenic substrates in the blood in the period between meals, we sought variations in their rate of conversion to glucose as potential correlates of satiety and hunger. Finally, increase and decrease of glucose uptake by an insulin-sensitive region of the hypothalamus has been proposed as a satiety and hunger signal [33,34]. Furthermore, although it remains to be shown that they function in behavioural rather than autonomic and endocrine control [23,53], some hypothalamic neurones have discharge rates which appear to relate directly to their rate of glucose oxidation [39]. We therefore included in the present experiments a search for a brain region with a rate of glucose uptake and catabolism which was transiently raised shortly after a meal. To be physiologically relevant we used a test meal of maintenance diet, given under conditions close to ad lib intake - i.e. within a normal hunger-satiety cycle. We also used

N EWMA N AND B()()'IH

radioisotopic techniques to provide estimate~ of metabolic fluxes and not merely isotope concentrations which by themselves are not biochemically interpret~ble. M [THOD

Rats, Housing and Diets Male hooded rats bred in the Department's Animal Laboratory were used in all experiments. They were adapted for at least two weeks to one of two rooms with opposite cycles of 12 hr light and 12 hr dark. The rooms were air-conditioned and maintained at 21-24°C. Before testing, the rats were housed in individual cages for at least one week. Each animal was fed and watered ad lib until the day of experiment. The maintenance diet was a laboratory chow (Modified Breeding Diet Cubes, Pilsbury's Ltd., Birmingham) which contained, on a dry weight basis, 48.6% carbohydrate, 3.8% nitrogen and 15.4 MJ/kg metabolisable energy. Pre-weighed amounts of the same chow blocks were used for test meals in determinations of gastric emptying, blood chemistry and tissue glucose metabolism.

Gastric Emptying Groups of rats were given test meals at selected times of day after periods of chow deprivation that had been found to be sufficient but no longer than necessary to ensure virtually empty stomachs. Except for one condition noted in Table 1, stomach contents in rats killed at the time the test meal was due were in the range of 0.01-0.15 g dry weight. The chow deprivation periods for the various test times were as follows (Dk and Lt refer to hours after the start of the dark and light periods, respectively): Test meals at Dk3 and Dk8: deprived for 1.5 hr, fed 1.5 g and deprived for a further 2.5 hr; Test meals at D k l l , Lt2 and L t l h deprived for 5 hr; Test meals at Lt5 and Lt8: deprived for 8 hr. The test meal was placed on the floor of the cage. Each rat's food intake was measured by difference in weights of chow available to the rat before and after the test meal, correcting for food lost through the bottom of the cage. At one of the predetermined times after the test meal, the rat was killed by cervical dislocation. Clamps were then placed at the base of the oesophagus, immediately below the pylorus and at the end of the small intestine. The contents of the stomach and of the small intestine in three sections of equal length were washed into vials with distilled water and their dry weight determined by heating to constant weight to 100°C. The carbohydrate in the samples of gut contents was solubilised by boiling with hydrochloric acid and hydrolysed to glucose with amyloglucosidase. Glucose was estimated with hexokinase and glucose-6-phosphate dehydrogenase [3]. Nitrogen was determined by Kjeldahl oxidation followed by Nesslerisation [58].

Systematic Metabolites For the determination of liver glycogen, the entire liver was quickly frozen in liquid nitrogen and ground into small pieces with a mortar and pestle kept cold with liquid nitrogen. The liver pieces were immediately added to a weighed tube of hot K O H (30 g/100 ml). The glycogen was purified by precipitation with ethanol and Na2SO4 [26] and, after suitable dilution, triplicate aliquots were measured by the phenolsulphuric acid procedure [12,36]. A standard curve was pre-

GASTRIC EMPTYING, METABOLISM AND F E E D I N G pared for each set of assays using glycogen standardised by the determination of glucose after hydrolysis with amyloglucosidase [3]. For the determination of blood metabolites, blood from the neck was collected in a beaker containing a few mg of heparin. An aliquot of the blood (about 1 ml) was immediately added to a weighed amount of 1 M perchloric acid (3 ml). After neutralisation with KOH, glucose, alanine, lactate and glycerol were determined enzymicaUy - - glucose by the hexokinase glucose-6-phosphate dehydrogenase method [30], alanine using its dehydrogenase [59], and lactate and glycerol using kits (Boehringer Corporation, London). In other experiments (as stated in Results), amino acid concentrations in whole blood supernatant were measured by standardised photometry of the ninhydrin reaction after automated chromatography using Li buffers.

931

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NOT FED 500

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Hepatic Gluconeogenesis Hepatic gluconeogenesis was estimated from the incorporation of radioactivity from L-(U-14C)-alanine into blood glucose [16,26]. This method uses about 1 mmol of cold alanine per rat, which is insufficient to promote glycogen synthesis but is considered sufficient to minimize any differences in specific activity at the site of glucose synthesis that might arise from dilution of labelled precursors by endogenous metabolites and also to reduce the effect of changes in glucose utilization, including recycling via the Cori cycle. Rats were injected intraperitoneally with L-(U-~4C)alanine (20/zCi/kg and 4 mmol/kg) in water (8 ml/kg). After 15 min the rats were stunned and decapitated, and blood from the neck was collected in a beaker containing a few mg of heparin. The blood was then immediately added to a weighed amount of perchloric acid (1 M; about 1 ml/ml blood). After centrifugation, the supernatant was neutralised with potassium hydroxide (4 M). The potassium perchlorate precipitate was removed by centrifugation and the solution stored at -20°C. A neutral fraction was prepared as described for extracts of the brain (below). The neutral fraction was examined by descending chromatography on Whatman No. 1 paper using propan-l-ol/ethyl acetate/water (7:1:2, by vol) as solvent. Standard sugars were applied as markers and were detected by the silver nitrate method [54] using sodium thiosulphate (10%, w/v) to fix the papers. Radioactivity on the paper was detected with a radiochromatogram scanner.

Brain Glucose Metabolism Rats were injected subcutaneously with U-~4C-glucose (287 mCi/mmol; 4 ml/kg). (Intraperitoneal administration was not used as at least a proportion of any compound administered by that route passes through the liver before reaching other organs [31 ], which results in a reduction of the specific activity of glucose at the brain. Compounds administered by the subcutaneous route are distributed to the entire body via lymphatic drainage into vena cava.) After the planned incorporation period, the rat was killed by immersion in liquid nitrogen and was stored in dry ice until dissection. The brain and a sample of blood from the heart were removed without allowing the carcass to thaw. Four regions were dissected from the brain at about - 10° to -5°C. Transverse cuts were made at the level of the optic chiasma and immediately posterior to the mammillary bodies. The "hypothalamus" sample was then removed by cutting horizontally 2 mm below the ventral surface, taking

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FIG. 1. Specific activity of glucose in blood, brain and liver after injection of U-14Cglucose (100/zmole/kg). Rats of body weight 230360 g were deprived of food for 5 hr in the light and then either not fed or allowed to re-feed for 20 min. There were 6 rats per mean for the blood and brain and 3 for the liver.

the anterior commissure as a reference, and then cutting longitudinally through the cerebral peduncles. The tissue left by the longitudinal cuts was combined to give the "amygdala" sample. The cortex was separated from the "thalamus" sample by the lateral ventricle. The "forebrain" region consisted of all the brain tissue anterior to the transverse cut at the optic chiasma. The tissue was extracted with HCl-methanol at between - 2 0 and -30°C [30]. An aliquot of the HCI extract was removed for the fluorometric determination of haem protein [37] using haemoglobin solutions standardized by conversion to cyanmethaemoglobin with Drabkin's Reagent [11]; brain glucose data were then corrected for brain blood content. Glucose, lactate and glutamate were also estimated fluorometrically [30J----glucose by the hexokinase method and lactate and glutamate by enzymic conversion to pyruvate and 2-oxoglutarate, respectively. For the determination of the specific activity of glucose, neutralized extract (1 ml) was passed through a column of Dowex 50 (H + form, 8% cross-linked, 200-400 mesh; 4 × 0.9 cm) and then a column of Dowex 1 (formate form, 10% cross-linked, 200-400 mesh; 4.1 cm). Neutral compounds were eluted with water (4 ml) and the specific activity of

932

NEWMAN AND B()Ol'I~t washed with water (3 ml) and amino acids eluted with ammonia (6 ml, 1 M). The ammonia solution was evaporated to dryness at 40°C in a Vortex evaporator I Buchler) and the dried extract was dissolved in water ~1 ml). This was adjusted to pH 8-9 and added to a Dowex 1 column (acetate form, 2 x 0.9 cm). The column was washed with water (5 ml) and glutamate eluted with acetic acid (8 ml, 0. I M). Glucose uptake was estimated as the ratio of the activity of the brain acid-soluble fraction (dpm/g) to the specific activity of the glucose from blood (dpm/nmol). In the main experiments, the specific activities of glucose in blood and brain were estimated 5 min after administration of U"C-glucose. Thus the calculated uptake values were overestimates because in pilot experiments the 5-min values were found to be the maximum values of specific activity in blood (Fig. 1). However, as both deprived and fed rats showed similar patterns of increase of glucose specific activity, these calculations can be used for comparisons between the two conditions.

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glucose determined by the preparation of glucose-6phosphate [20]. The specific activity of lactate was determined after the preparation of the 2,4-dinitrophenylhydrazone [51]. For determination of its specific activity, glutamate was isolated by a modification of Gaitonde's method [18]. Neutralized extract (2 ml) was adjusted to pH 8-9 and added to a Dowex 50 column (H ÷ form, 2 x 0.9 cm). The column was

Gastrointestinal Contents Dry weights of gastric contents declined rapidly after a meal at Dk3 (Fig. 2). Despite this, intestinal contents at the intervals tested varied little if at all under these conditions of minimal food deprivation (Fig. 2), in contrast to refeeding after extended deprivation [57]. The pattern of gastric emptying at all times of day studied was assessed by least squares linear regression analysis [13] of the series of groups of rats at each time point after a test meal. As it was not possible to achieve exactly the same meal size for each rat because of variable spillage, the gastric contents were individually standardised by dividing the stomach content found in a rat by the meal size for that rat. A time point for a given condition of meal size and time of day was not included in the regression for that condition if one or more rats in that group were found to have empty stomachs: this avoided artefactual curvature of the group emptying pattern at late time points. Linear regressions were carried out on untransformed data (linear emptying), or on logarithm (exponential emptying [22]), square root [21,22] and reciprocal of the cube root [52] of fractional contents, and on logarithm of time (logarithmic emptying). The function which gives the best fit to the data has the highest F-ratio in the analysis of variance of the regression (Table 1). No function fitted markedly better than the untransformed data, and so linear emptying rates are used in subsequent figures here. The square root transformation [7, 8, 10] was the best fit about as frequently as the linear emptying, and either function was a much better fit to the data than any of the other three transformations. The gastric empyting rate was not statistically dependent (,o>0.05) [13] on body weight or meal size, within the ranges investigated (Fig. 3). At each of seven times around the 12 h r - 12 hr dark-light cycle, test meals of about 2 g were given after the minimal deprivation to empty the stomach (Method) and the contents of the stomach determined after 0.5 hr intervals (Table 1). The gastric emptying rate showed a nycthemerat rhythm, with a peak at the start of the dark period and a nadir at the start of the light period (Fig. 4). This corresponds to periods of high and low meal frequency respectively [29]. These data for the light period were obtained from rats housed in one

GASTRIC EMPTYING, METABOLISM AND F E E D I N G

933

TABLE 1 LINEAR REGRESSIONFITS TO GASTRICEMPTYINGPATTERN Time of meal (hr after start of light period)

Mid-point of body weight range* (g)

Mean meal size (g)

Number of rats

Duration of emptying considered (hr)

Q

25 5 8 11 15 20 23

255 255 255 255 255 255 255

1.8 2.1 1.8 1.9 2.0 2.0 1.9

12 21 13 16 16 16 14

4 3 3 3 2 3 4

12.7 147.5 67.3 79.8 70.6 76.5 84.2

14.8 174,1 42,9 54.6 47,1 64.5 133.7

13.8 125.7 6.8 6.7 3.7 20.0 126.5

15.0 154.0 14.3 17.9 11.0 32.8 155.0

15 15 15 15 15

255 255 175 395 325

2.7 3.6 2.1 2.1 2.7

16 24 12 15 13

3 3.5 3 3 2

119.6 208.1 93.7 32.7 53.7

159,9 138.1 157,5 32.9 44.4

35.5 19.0 19.3 13.6 28.8

68.6 43.9 42.4 21.5 34.5

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*Range of body weights in each group was 30 g. tHighest F-ratio at a particular time is the best fit to the data. Q is percentage emptied from the stomach. SAt this time rats killed instead of fed had gastric contents of 0.5-1.0 g dry weight. Baseline gastric contents at all other times was 0.1-0.15 g dry weight. §These rats were housed in the room normally used for light period measurements.

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room and the data for the dark period from rats housed in another room. However, the rate differences were atrributable to lighting phase, not rooms: when the emptying rate of rats in the room normally used for light-period measurements was measured during the dark period, the rate observed (0.55 _+ 0.07 g/hr) was not significantly different from that of rats tested in the dark in the room normally used for dark period measurements (0.72 _+ 0.09 g/hr). Carbohydrate and protein content. The percentage of carbohydrate in the gastric and intestinal contents varied little if at all with time after a meal, although almost all of the carbohydrate had been absorbed by the time the chyme had left the small intestine (Fig. 5). The dry weight of intestine contents had also remained constant from 30 rain after the meal until the stomach was practically empty (Fig. 2). Therefore, at the times sampled, the rate of emptying of carbohydrate from the stomach was matched by the rate of carbohydrate absorption from the small intestine. This agrees with previous conclusions, drawn from experiments under different conditions, that the rate of absorption of carbohydrate depends on the rate at which it passes from the stomach to the duodenum [40,41]. Although dietary protein content of the gut was not separately measured, the nitrogen contents of the intestine (Fig. 5) suggested that secreted and shed protein approximately replaced absorbed dietary amino acids [41]; presumably, amino acid absorption was occurring approximately in parallel with glucose absorption. The peripheral cholinolytic drug, atropine methyl nitrate, greatly inhibited gastric emptying in the dark period (Fig. 6). This is consistent with the possibility, among others, that the observed rapid gastric emptying of solids by night in the rat depends on vagal cholinergic facilitation.

934

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7 11 15 19 23 TIME AFTER START OF LIGHT PERIOD (hr) FIG. 4. Nycthemeral variation of gastric emptying rate. Width of column represents period of emptying measured using 12-24 rats, given test meals of group mean sizes 1.8--2.1 g in both light and dark (first seven rows of Table 1" one-way ANOVA p<0.02).

FIG. 5. Carbohydrate (©) and nitrogen (0) proportions (%) in the contents of stomach and small intestine of rats (240-340 g) after a test meal of average size 2.7 g dry weight at 3 hr after the start of the dark period. Values are means _+ SD for 3-7 rats. Chow diet was 48.6% carbohydrate and 3.8%, nitrogen.

Injection

Blood Glucose and Liver G4'cogen No statistically reliable rise in the relatively low hepatic glycogen content was detected following a 3-g meal of chow eaten under close to ad lib conditions near the start of the dark period (Dk3, Method). Liver glycogen did not decline substantially up to 3 hr after the meal (Table 2, Group A). As this meal was of fixed size and possibly not satiating, the experiment was repeated allowing the rats to feed ad lib for 30 min after the deprivation for 2.5 hr scheduled for tests at Dk 3. Again, no marked variations in hepatic glycogen content were seen for at least 3 hr (Table 2, Group B). An overall rise in hepatic glycogen content throughout the dark phase may compensate at the time of these measurements for any tendency for liver glycogen to fall post-digestively [27]. Postprandial hyperglycaemia in rats fed 3 g under these conditions proved to be too brief or variable in timing to be detectable at the times sampled by these groups post mortem methods (Table 2: see also Tables 4, 6 and 8). In a separate experiment, rats under the same conditions (except that food was not removed 30 min after the start of the meal) were observed until they started another meal. Consistently with others' findings for the dark period [29], the subsequent meal was found to occur within 60-120 min after the start of the test meal. Thus a decline in liver glyco-

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FIG. 6. Effect of atropine methyl nitrate (10 mg/kg body weight) injected subcutaneously on gastric emptying after a test meal of 2 g (dry weight) at 3 hr after the start of the dark period.

Fen cannot be the general condition for starting a meal [9, 43, 56].

Gluconeogenic Capacity and Substrate Concentrations Hepatic gluconeogenic capacity was decreased during the

GASTRIC EMPTYING, METABOLISM TABLE 2

HEPATIC GLYCOGEN CONTENTS AND BLOOD GLUCOSE CONCENTRATIONS IN RATS FED EARLY IN THE DARK PERIOD Time after testmeal start (min) 0 30 60 90 120 180 240

Hepatic glycogen (% fresh weight) Group A* 2.1 3.0 1.5 2.3 2.3 2.4 1.9

+_ 1.1 _+ 1.8 + 0.7 _+ 0.8 -+ 0.8 ___ 1.0 _+ 1.2

Blood glucose (it mol/g)

Group Bt

(4) (6) (6) (6) (7) (7) (5)

2.0 1.7 1.5 1.2 1.3 1.7 1.1

_+ 0.8 + 0.4 _+ 0.1 +- 0.3 _+ 0.4 _+ 0.2

935

AND FEEDING

Group B t

(3) (3) (3) (3) (3) (3) (2)

5.3 4.6 5.4 4.8 4.5 4.6 4.4

- 1.5 (5) -+ 0.8 (4) _+ 0.9 (4) _+ 1.0 (4) _ 0.5 (4) _+ 1.0 (4) (2)

TABLE 3 GLUCONEOGENIC CAPACITY ESTIMATES IN RATS CLOSE TO AD LIB* IN DARK PERIOD

Time of start of incorporationt Not fed 30 60 90 120 180 240

Blood glucose activity (dpm/mg) 4.4 2.9 2.8 3.7 3.7 3.7 5.1

Blood alanine specific activity (dpm/mmole)

_+ 0.6 -+ 0.4 _+ 0.1 -+ 0.2 _+ 0.1 -+ 1.3

4.5 4.0 3.8 4.9 4.4 4.4 4.5

_+ 0.9 _+ 1.0 _+ 0.6 _+ 1.9 _+ 0.5 _+ 0.9

Glucose Alanine 1.05 0.76 0.76 0.87 0.84 0.87 1.12

+_ 0.29 +- 0.19 _+ 0.12 _+ 0.43 _ 0.09 _+ 0.34

Values are means _+ S.D. (number of rats). *Fed the fixed amount of 3 g (all eaten) at Dk3 after the standard pre-test feeding schedule (see METHOD). t F e d for 30 min at Dk3.

Values are means _+ SD for 3 rats. *Fed for 30 min after the scheduled feeding of the Dk3 condition of the gastric emptying Method (q.v.). tTimes after start of Dk3 meal at which L-(U-14C)-alanine injected.

TABLE 4 SUBSTRATES IN THE BLOOD ESTIMATED ENZYMICALLY

TABLE 5 BLOOD CONCENTRATIONS OF SUBSTRATES ESTIMATED ENZYMICALLY

Time (rain) after start of meal* -30 0 30 60 90 120 180 240

Gluconeogenic substrates (/~mob'g) Glucose (/~mol/g) 4.0 4.8 4.2 4.3 4.2 4.1 4.4 4.4

_+ 0.3 _+ 0.5 _+ 0.4 _+ 0.3 _+ 0.4 _+ 0.4 _+ 0.2 _+ 0.3

Alanine 0.24 0.21 0.30 0.25 0.24 0.23 0.21 0.20

___0.02 - 0.04 -+ 0.04 -+ 0.03 - 0.03 --- 0.02 - 0.03 -+ 0.02

Glycerol 0.14 0.14 0.14 0.14 0.12 0.13 0.13 0.13

_+ 0.02 _+ 0.02 --- 0.02 _+ 0.01 -+ 0.03 -+ 0.02 - 0.02 -+ 0.02

Glucose

Alanine

Glycerol

Lactate

2.5 _+ 0.3

0.12 _+ 0.02

0.10 _+ 0.02

1.40 _+ 0.35

3.4 _+ 0.3

0.23 _ 0.03

0.06 _+ 0.01

1.17 _+ 0.40

Lactate 1.17 1.14 1.20 1.28 1.15 1.44 1.05 1.06

- 0.32 _+ 0.30 _+ 0.36 -+ 0.32 _+ 0.39 _+ 0.42 _+ 0.39 _+ 0.36

Values are means --- SD for 4 rats. *Rats were allowed to eat for 30 min in the dark (at Dk3 with deprivation period as in gastric emptying Methods).

p e r i o d t h a t e n e r g y w a s b e i n g a b s o r b e d f r o m the gut (Table 3: g l u c o s e / a l a n i n e ratio v a r i e d o v e r 6 g r o u p s o f 3 rats, p <0.05). G l u c o n e o g e n i c s u b s t r a t e s (alanine, lactate, glycerol), like g l u c o s e itself, s h o w e d m o d e s t c h a n g e s or n o c h a n g e in conc e n t r a t i o n in t h e b l o o d o v e r this p e r i o d (Table 4 a n d Fig. 7). This w a s c o n s i s t e n t w i t h m o d e s t d i f f e r e n c e s in a l a n i n e a n d o t h e r a m i n o acids b e t w e e n rats s a m p l e d at r a n d o m f r o m g r o u p s feeding ad lib a n d g r o u p s s t a r v e d for 24 h r (Table 5 a n d Fig. 7). T h u s , t h e i n c r e a s e in g l u c o n e o g e n i c c a p a c i t y following t h e slowing o f a b s o r p t i o n m a y b e partly m e d i a t e d h o r m o n a l l y [46].

Brain Glucose Uptake and Catabolism Shortly After Meals I n t h e light p e r i o d , b l o o d g l u c o s e c o n c e n t r a t i o n a n d b r a i n g l u c o s e u p t a k e a n d c a t a b o l i s m did n o t a p p e a r to b e i n c r e a s e d at 20-25 rain a f t e r a m e a l (Table 6). H o w e v e r , at this time a f t e r a m e a l in t h e dark, the r e s u l t s o f T a b l e 6 s h o w e d b o t h

Starved for 24 hr Fed ad lib

Values are means _+ SD for 4 rats, in/zmoles/g of blood. The rats were killed 3 hr after the start of the dark period.

a n h y p e r g l y c a e m i a a n d a n i n c r e a s e d rate o f t r a n s p o r t o f gluc o s e into b r a i n tissue. Signs o f i n c r e a s e d c e r e b r a l glycolysis w e r e a p p a r e n t b u t t h e y w e r e n o t c a r r i e d t h r o u g h to e i t h e r the a n a e r o b i c or t h e t r i c a r b o x y l i c acid cycle i n t e r m e d i a t e m e a s u r e d (Table 6). I n c o r p o r a t i o n into b r a i n g l y c o g e n was not d e t e c t a b l e o v e r the 5-min p e r i o d used. T h e i n c r e a s e s in b r a i n g l u c o s e u p t a k e a n d in c a t a b o l i s m to acid-soluble interm e d i a t e s were n o t localized to a n y o n e o f the four brain r e g i o n s s a m p l e d (Table 7). The observations of Tables 6 and 7 were not replicated by a s t u d y (Table 8) u s i n g less i n j e c t e d r a d i o a c t i v i t y (50/xCi/kg). T h e i n c r e a s e in b r a i n g l u c o s e u p t a k e in the d a r k period, like t h e p o s t p r a n d i a l h y p e r g l y c a e m i a , m a y v a r y in t i m i n g or m a y n o t e v e n o c c u r a f t e r s o m e m e a l s , d e p e n d i n g o n the effects of the meal o n the earliest s t r e s s o f digestion a n d a b s o r p t i o n (Tables 2 a n d 4). DISCUSSION T h e rat t a k i n g a n o r m a l l y sized meal o f a s t a n d a r d laborat o r y diet u n d e r close to ad lib c o n d i t i o n s s h o w e d a n u n e x p e c t e d l y m o d e s t d e c e l e r a t i o n o f gastric e v a c u a t i o n up to the time at w h i c h the s t o m a c h w a s a l m o s t e m p t y . T a k e n w i t h the o b s e r v e d c o n s t a n c y o f intestinal c o n t e n t s , this implies t h a t the r a t e s o f a b s o r p t i o n o f c a r b o h y d r a t e s a n d o t h e r n u t r i e n t s v a r y r a t h e r little b e t w e e n m e a l s o n l a b o r a t o r y c h o w ad lib.

NEWMAN AND BOO'IH

936

0.5

__0-Z, o') 0~ o

E

s~

73.

zO-3

r'"

;~

0

T

rY z w

o

Z 0 0 D 0 0 .._1

s

0.2

m 0.1

Ad libitum

0

1

2

3

~

24hrStarved

TIME SINCE FEEDING (hr) FIG. 7. Concentrations of free amino acids in whole blood at various times since feeding, measured chromatographically in 2-4 rats per mean (vertical bars: SDs). Vertical dashes mark the mean time of the end of the test meal. Hatching covers the period in which the next spontaneous meal would have occurred if food had been left in the cage. Glycine and serine showed patterns similar to alanine but at slightly lower concentrations. Glutamate, glutamine and asparagine were not resolved because of an interfering unknown in these assays.

The possibility of a brief burst of rapid absorption during or shortly after the meal could not be excluded, however, because of limitations of the only practicable method of intermittently timed post mortem analyses of groups of animals. A brief initial phase of rapid gastric emptying, a long period of relatively constant rate, and finally a slowing of gastric emptying as the stomach becomes almost empty, have been observed in man when composite [24] or ordinary [32] meals of normal size are studied carefully. Even clearer and more important was our detailed confirmation for maintenance chow of earlier indications [9,10] that the gastric emptying function is markedly steeper in the dark phase than in the light phase of the lighting cycle. Considerable metabolic, hormonal and behavioural consequences of this nycthemeral rhythm are to be expected. The faster gastric emptying at night coincides with the rat's period for net deposition of fat, much greater food intake and somewhat greater metabolic rate [27,29]. Our results show that the gastric rhythm is a primary factor in these cycles. In

particular, the gastric rhythm is not caused by the feeding rhythm. The present experiments used test meals of fixed size on a recently empty stomach. Thus, the observed differences in emptying rate between day and night could not have resulted merely from differences in amount of food recently eaten or remaining in the gut. The gastric emptying rhythm is not directly driven by the pattern of lighting either, for emptying rate increased some hours before the lights were switched off. There are nycthemeral rhythms in intestinal disaccharidase activity and glucose transport [14, 15, 50] which also anticipate food intake and are not a direct consequence of it [45]. The enzyme rhythms are unlikely to cause the emptying rhythm, at least via the duodenal osmoreceptor inhibition of gastric emptying 122], because at night (when emptying is faster) disaccharidase activity is higher and would if anything inhibit emptying by delivering monosaccharide faster to the osmoreceptor. Gastric emptying, intestinal digestion and transport capacities and endocrine responses [47] may all be facilitated in parallel, perhaps via the autonomic nervous system, to anticipate the fast processing of nutrients by night in the rat (or conversely inhibited by day for slow processing). Both the steep gastric emptying function and a stronger insulin secretory response appear from observations [27,28] and from calculations [8,10] to be causal preconditions for the frequent meals and net lipogenesis which are seen by night at the intact rat, and are seen in the day as well as in the night in rats with lesions in the ventromedial hypothalamus [8, 10, 23, 28]. That is, the metabolic consequences of rapid emptying release hunger sooner and so increase meal frequency. Also, rapid emptying and facilitated lipogenesis may reduce the satiating power of a given size of meal and so increase meal sizes somewhat, both normally in the dark phase, and pathologically during the hyperphagia which is secondary to hyperinsulinaemia [8,23]. One implication our results have for procedures commonly used in biochemistry laboratories is that the "fed rat" (taken from the ad lib feeding condition without regard to the time it last took a meal) may be metabolically a somewhat more homogeneous preparation than might have been the case with a strongly decelerating absorption function. On the other hand, our results emphasise what has also been apparent from other work, that the metabolic and hormonal status of rats taken from the colony in daylight cannot be generalised to the dark period nor extrapolated to estimate net effects over 24 hr in accounting for long term effects on body composition etc. The results of blood and tissue analyses supported the overall impression that, in the laboratory rat which has adapted its meal pattern to continuous access to a single maintenance diet, internal controls of gastric emptying, like those of intermediary metabolism, contribute considerable stabilization to the supply of energy to the brain and other working tissues [5, 8, 10]. The stomach is relatively repleted and depleted of food from meal to meal but little else is. In particular, variations in glucose supply to the brain between meals may be reduced or even eliminated by the observed decrease in gluconeogenesis until the time when absorption begins to slow appreciably. By the same token, the nature and origin of the substrates being oxidised by the liver varies with abundant or deficient energy absorption and might be signalled via hepatic afferents to the cerebral appetite system. In other words, changes in the rate of gluconeogenesis may provide the basis of a signal of the rate of satiating energy flow within the body [5, 7, 17]. Gluconeogenic sub-

GASTRIC EMPTYING, METABOLISM AND FEEDING

937

TABLE 6 CEREBRAL METABOLIC FLUXES WITHOUT FEEDING OR AFTER FEEDING IN LIGHT AND DARK PERIODS Light period*

Blood glucose (p,mol/g) Brain glucose uptake (p,mol/g/5 min) Conversion of brain glucose (/zmol/g/5 rain): to "metabolised glucose" to lactate to glutamate

Dark periodt

Not fed

Injected 20 min after start of feeding

Injected 25 min after start of feeding

Injected when feeding started

Injected 20 min after meal end

5.6 ± 0.I (4)

5.3 ± 0.2 (4)

5.4 ± 0.3 (5)

5.7

± 0.1 (3)

5.5

± 0.7 (3)

3.3 ± 0.5 (4)

3.2 ± 0.3 (4)

3.6 ± 0.8 (5)

2.8

± 0.8 (3)

4.8

± 0.6/3)

1.7 ± 0.2 (4) 0.4 ± 0.2 (4) --

2.0 ± 0.2 (4) 0.6 _+ 0.1 (4) --

2.9 ± 0.7 (5) 0.6 ± 0,3 (5) --

3.7 ± 1,5 (3) 1.12 ± 0.37 (3) 0.75 ± 0.17 (3)

4.4 ± 2.7 (3) 0.72 ± 0.24 (3) 0.75 ± 0.47 (3)

Values are means - SD (numbers of rats). The value of each rat was the mean of 4 brain regional values. *Body weights 300-370 g, deprived for 5 hours; fed groups given ad lib access to maintenance chow. tBody weight 200-230 g, not deprived of food until injection of U-~4C-glucose, timed according to visually observed start of a spontaneous meal.

TABLE 7 REGIONAL METABOLIC FLUXES WITHOUT FEEDING OR AFTER FEEDING IN DARK PERIOD Liver Tissue uptake of glucose Glucose to catabolites Glucose to lactate Glucose to glutamate

Unfed Fed Unfed Fed Unfed Fed Unfed Fed

7.1 9.7 3.2 3.2 0.32 0.39

___ 0.6 -4- 0.1 _ 0.7 ± 0.18 ---

Hypothalamus 2.7 4.6 4.0 4.5 1.23 0.77 0.54 0.60

-+ 1.2 ± 0.7 _+ 2.9 ± 3.4 ± 0.23 ± 0.37 ± 0.25 ± 0.46

Amygdala 2.8 4.3 2.3 4.2 0.67 0.66 0.34 0.79

± 0.4 _+ 0.8 -+ 1.3 ± 4.0 ± 0.63 ± 0.47 ± 0.13 ± 0.78

Thalamus 2.7 5.2 5.0 3.7 1.77 0.63 1.45 0.61

_+ 1.2 ± 0.7 ± 3.9 ± 1.2 ± 1.75 ± 0.11 ± 0.79 ± 0.13

Forebrain 3.2 5.1 3.6 5.2 0.82 0.84 0.69 1.00

± + ± ± ± ± ± ±

1.0 0.4 1.1 2.7 0.27 0.33 0.08 0.56

Values are means _+ SD (in tzmoles/g/5 min of incorporation in the dark-phase rats of Table 6.

TABLE 8 BLOOD GLUCOSE CONCENTRATION AND BRAIN GLUCOSE UPTAKE IN THE DARK PERIOD IN FED RATS Injection at meal start Blood glucose (Ixmol/g) Brain uptake (/zmol/g/5 min)

5-min incorporation period started: after meal end At I0 min

At 20 min

At 40 min

6.3 _+ 0.4

5.7 -+ 0.7

6.2 _ 0.6

4.8 ± 1.1

3.3 _+ 0.6

3.4 ± 1.8

2.8 _+ 0.5

2.6 _+ 0.6

Values are means ± SD for 3 rats (body weights 270-310 g) fed at Dk3 after the standard deprivation-feeding schedule (METHOD).

strates r e p r e s e n t m a j o r e n e r g y s o u r c e s distinct f r o m intestinal a b s o r p t i o n , i.e. glycerol (from fats), certain a m i n o acids s u c h as alanine (released after r e - s y n t h e s i s in muscle) and lactate (greatly i n c r e a s e d after exercise). E f f e c t s o n the t r a n s p o r t and utilization o f t h e s e s u b s t r a t e s , and h o r m o n a l r e s p o n s e s to feeding and in turn to s t a r v a t i o n , p r e s u m a b l y c a u s e the r e d u c t i o n in g l u c o n e o g e n e s i s after a meal and its rise as a n o t h e r meal b e c o m e s d u e , w i t h a still f u r t h e r rise as fasting e n s u e s . Only f u r t h e r w o r k on s u b s t r a t e fluxes and p a t h w a y capacities within t i s s u e s can c o n f i r m o r refute this h y p o t h e s i s o f an influence o n h u n g e r and satiety. A n alternative o r c o m p l e m e n t a r y i n t e r p r e t a t i o n is that the timing o f m e a l s a n d their sizes (to the e x t e n t that t h e y are not r e s p o n s e s to r a n d o m o r s y s t e m a t i c e v e n t s u n r e l a t e d to nutrition) are d e t e r m i n e d by a n t i c i p a t o r y a v o i d a n c e o r attenuation o f e v e n t s that are relatively unusual in the freely feeding o r g a n i s m , e.g. a p r e - p r a n d i a l failure o f a b s o r p t i o n bec a u s e the s t o m a c h has b e c o m e a l m o s t e m p t y o r a postprandial b u r s t o f initially u n c o n t r o l l e d gastric e m p t y i n g be-

938

NEWMAN AND Bf)OTH

cause of a depleted d u o d e n u m or e x c e s s i v e gastric filling - events which cannot be occurring frequently by night or day, as may be seen by comparing our gastric emptying data with the meal sizes and meal-to-meal intervals o b s e r v e d in freely fed rats in many laboratories. For example, meal initiation and termination ad lib could be controlled by gastric cues appropriate to the diet and time of day [7,8], associatively conditioned [6,7] by the u n e x p e c t e d metabolic or hormonal c o n s e q u e n c e s of the occasional early or late, large or small meal. Such transient and variable effects will be extremely difficult to measure by available methods, because of problems of controlling the metabolic status of a freely feeding rat as it c o m e s to a normal meal and because of limits to the quantification of spontaneously occurring biochemical processes which change in rate within minutes. On-line blood metabolic and h o r m o n e levels can be m e a s u r e d o v e r the required time course [9] but such data permit no conclusions as

to events at d e t e c t o r or receptor sites in tissues. If some transients affect brain metabolism, as our data indicate, the relevant region of brain tissue may be a few mg at most. The 2-deoxyglucose uptake method [48] may have the required anatomical resolution b u t - - e v e n if quantitatively valid, which has been questioned l l 9 ] - - r e q u i r e s 30 or 45 min of incorporation, is subject to competition from the transport of glucose itself, and cannot measure the relevant glucose uptake caused by glucose availability and insulin levels as opposed to neuronal demands. Therefore it may well be that the behavioural effects of controlled and measured disturbances, within the range of ordinary variations of dietary composition, ecological demand and individual dynamics, will tell us more about the biochemistry of appetite in the foreseeable future than any available techniques for measuring correlates o f meal initiation and termination in tissue biochemistry.

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