Cyclic estradiol treatment normalizes body weight and restores physiological patterns of spontaneous feeding and sexual receptivity in ovariectomized rats

Hypothalamic-pituitary-gonadal axis function strongly influences feeding and body weight in cycling females in many species. To test the sufficiency of cyclic variations in plasma estradiol to reproduce normal patterns of spontaneous feeding, food intake, and body weight, ovariectomized Long-Evans rats were subcutaneously injected every fourth day with 2 microg estradiol benzoate or with the oil vehicle alone. Cyclic estradiol treatment completely normalized the trajectory of body weight gain and total food intake through seven treatment cycles. The hyperphagia of ovariectomized rats was expressed as an increase in spontaneous meal size. Meal frequency decreased, but not enough to compensate for the increase in meal size. Estradiol treatment normalized both parameters. In addition, cyclic estradiol treatment produced a further phasic decrease in meal size (and increase in meal frequency) and a decrease in food intake during the second night after injection. This phasic change is similar to the feeding changes occurring during estrus in intact rats. Sexual receptivity was measured during the eighth estradiol treatment cycle, 4 h after injection of 0.5 mg progesterone. Lordosis scores at the time of the treatment cycle modeling estrus were maximal, and scores at the time modeling diestrus were slightly increased over those of rats that did not receive estradiol. Finally, plasma estradiol levels, measured during the ninth treatment cycle, revealed a near-normal cyclic pattern of plasma estradiol levels. These results provide the first demonstration that the induction of a cyclic, near-physiological pattern of plasma estradiol is sufficient to maintain normal levels of body weight, spontaneous feeding patterns, total food intake, and (together with progesterone) sexual receptivity in ovariectomized rats.     

Estradiol treatment increases CCK-induced c-Fos expression in the brains of ovariectomized rats

The ovarian hormone estradiol reduces meal size and food intake in female rats, at least in part by increasing the satiating potency of CCK. Here we used c-Fos immunohistochemistry to determine whether estradiol increases CCK-induced neuronal activation in several brain regions implicated in the control of feeding. Because the adiposity signals leptin and insulin appear to control feeding in part by increasing the satiating potency of CCK, we also examined whether increased adiposity after ovariectomy influences estradiol's effects on CCK-induced c-Fos expression. Ovariectomized rats were injected subcutaneously with 10 microg 17beta-estradiol benzoate (estradiol) or vehicle once each on Monday and Tuesday for 1 wk (experiment 1) or for 5 wk (experiment 2). Two days after the final injection of estradiol or vehicle, rats were injected intraperitoneally with 4 microg/kg CCK in 1 ml/kg 0.9 M NaCl or with vehicle alone. Rats were perfused 60 min later, and brain tissue was collected and processed for c-Fos immunoreactivity. CCK induced c-Fos expression in the nucleus of the solitary tract (NTS), area postrema (AP), paraventricular nucleus of the hypothalamus (PVN), and central nucleus of the amygdala (CeA) in vehicle- and estradiol-treated ovariectomized rats. Estradiol treatment further increased this response in the caudal, subpostremal, and intermediate NTS, the PVN, and the CeA, but not in the rostral NTS or AP. This action of estradiol was very similar in rats tested before (experiment 1) and after (experiment 2) significant body weight gain, suggesting that adiposity does not modulate CCK-induced c-Fos expression or interact with estradiol's ability to modulate CCK-induced c-Fos expression. These findings suggest that estradiol inhibits meal size and food intake by increasing the central processing of the vagal CCK satiation signal.     

Physiological effect of circulating glucagon on the hepatic membrane potential

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane under various conditions. Here we investigated the physiological relevance of this effect by testing the influence of infusions of glucagon antiserum on the liver cell membrane potential in vivo. Intracellular microelectrode recordings of liver cells (up to 60/rat over 2 h) were done in anesthetized male rats. Livers were fixed in place, and recordings were done 10-30 min after intraperitoneal injections of glucagon or hepatic portal vein infusions of glucagon or specific polyclonal glucagon antibodies raised in rabbits. The isotonic lactose vehicle was used as a control for glucagon, and equal amounts of nonimmunized rabbit IgG were used as a control for glucagon antibodies. Intraperitoneal glucagon (400 microg/kg) hyperpolarized the liver cell membrane up to 12 mV, and intraportal glucagon (10 or 60 microg/kg) dose dependently hyperpolarized the liver cell membrane by 3-7 mV. Intraportal infusion of glucagon antiserum (in vitro binding capacity of 4 ng glucagon/rat) significantly depolarized the liver cell membrane by approximately 2.5 mV. The effects of both glucagon and glucagon antiserum reversed after 60-90 min. We conclude that glucagon is a physiologically important modulator of the liver cell membrane potential.     

Pancreatic glucagon fails to inhibit sham feeding in the rat

Rats were equipped with chronic gastric cannulas, and the effects of intraperitoneal injections of pancreatic glucagon on sham feeding, with cannulas open, and real feeding, with cannulas closed, were compared. Glucagon (100–2,500 μg/kg) suppressed cumulative food intake during real feeding tests 9–33%, but had no effect during sham feeding. Despite their increased food intakes, sham feeding rats took discrete meals terminated by the behavioral satiety sequence. In addition to not affecting total intake, glucagon failed to affect meal size, latency to rest, or intermeal interval during sham feeding. To investigate the possiblity that glucagon did not inhibit sham feeding because it did not elicit hyperglycemia, we measured hepatic vein blood glucose after glucagon injections in sham feeding rats: glucagon elicited marked hyperglycemia during sham feeding. Therefore, the absence of glucagon's satiety effect in sham feeding is not due to the lack of hepatic glycogenolysis and hyperglycemia. These results suggest that some mechanism other than hyperglycemia which normally accompanies food ingestion is necessary for glucagon to have a satiety effect.     

Differential impact of cocaine on meal patterns in female and male rats

Female rats, relative to males, exhibit greater behavioral activation to cocaine and other psychostimulants, but the effect of sex and the estrous cycle in modulating the hypophagic action of cocaine has not been evaluated. Meal patterns were recorded in automated food hoppers during the first 3 h of the dark phase in adult female and male rats after administration of ascending cocaine doses (0, 7.5, and 15 mg/kg cocaine, i.p.) on successive trials. Cocaine produced a greater suppression of feeding as well as a reduction in meal number over a 3 h test period in female rats during estrus, relative to that noted during diestrus. In contrast, during the 180 min test period, male rats showed minimal hypophagic responses to 7.5 or 15 mg/kg cocaine. These results extend the range of behavioral perturbations induced by cocaine that are modulated by sex and by the estrous cycle and are consistent with the notion that estradiol may modulate the neurochemical actions of cocaine.     

Estradiol, CCK and satiation.

Estradiol has long been known to inhibit feeding in animals, but the mechanism(s) mediating its effects have not been clear. Demonstrations that estradiol's feeding effects are expressed as decreases in meal size coupled with the emerging consensus that cholecystokinin (CCK) released from the small intestines during meals is a physiological negative-feedback signal controlling meal size (i.e. satiation) suggested a new approach to the problem of the mechanisms of estradiol's inhibitory effect on feeding. Progress on this approach is reviewed here. Experimental manipulations of exogenous and endogenous CCK and estradiol have produced converging evidence that estradiol cyclically increases the activity of the CCK satiation-signaling pathway so that meal size and food intake decrease during the ovulatory or estrus phase of the ovarian cycle. This is a striking example of the modulation of the operation of a control of meal size by the physiological context in which the meal occurs. Estradiol also produces a tonic decrease in meal size, but this apparently does not involve the CCK satiation-signaling pathway. Where and how estradiol acts to increase the potency of the CCK satiating-signaling pathway are not known. Several possible sites are suggested by the observations that estradiol treatment increases feeding- and CCK-induced expression of c-Fos in ovariectomized animals in brain areas including the nucleus tractus solitarius, paraventricular nucleus of the hypothalamus, and central nucleus of the amygdala. Tests with null mutation mice indicate that estrogen receptor-alpha is necessary for estradiol's feeding effects. Finally, the possibilities that estradiol exerts important influences on normal or disordered eating in women are discussed. It is concluded that estradiol exerts a biologically significant action on CCK satiation in animals. Further research to determine whether this action of estradiol has a role in the pathogenesis, course, or treatment of disordered eating in women is indicated.     

Estradiol treatment increases feeding-induced c-Fos expression in the brains of ovariectomized rats

The steroid hormone estradiol decreases meal size by increasing the potency of negative-feedback signals involved in meal termination. We used c-Fos immunohistochemistry, a marker of neuronal activation, to investigate the hypothesis that estradiol modulates the processing of feeding-induced negative-feedback signals within the nucleus of the solitary tract (NTS), the first central relay of the neuronal network controlling food intake, and within other brain regions related to the control of food intake. Chow-fed, ovariectomized rats were injected subcutaneously with 10 microg 17-beta estradiol benzoate or sesame oil vehicle on 2 consecutive days. Forty-eight hours after the second injections, 0, 5, or 10 ml of a familiar sweet milk diet were presented for 20 min at dark onset. Rats were perfused 100 min later, and brain tissue was collected and processed for c-Fos-like immunoreactivity. Feeding increased the number of c-Fos-positive cells in the NTS, the paraventricular nucleus of the hypothalamus (PVN), and the central nucleus of the amygdala (CeA) in oil-treated rats. Estradiol treatment further increased this response in the caudal, subpostremal, and intermediate NTS, which process negative-feedback satiation signals, but not in the rostral NTS, which processes positive-feedback gustatory signals controlling meal size. Estradiol treatment also increased feeding-induced c-Fos in the PVN and CeA. These results indicate that modest amounts of food increase neuronal activity within brain regions implicated in the control of meal size in ovariectomized rats and that estradiol treatment selectively increases this activation. They also suggest that estradiol decreases meal size by increasing feeding-related neuronal activity in multiple regions of the distributed neural network controlling meal size.     

Acute activation of ER alpha decreases food intake, meal size, and body weight in ovariectomized rats

Estradiol exerts many of its actions by coupling with two nuclear estrogen receptor (ER) proteins, ER alpha, and ER beta. While the acute, anorexigenic effect of estradiol appears to involve such a mechanism, the relative contributions of ERalpha and ERbeta are equivocal. To address this problem, food intake was monitored in ovariectomized (OVX) rats following acute administration of a selective ER alpha agonist (4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol, PPT; dose range = 0-200 microg), a selective ER beta agonist (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN; dose range = 0-600 microg), and a physiological (4 microg) dose of estradiol benzoate (EB). While PPT-treated rats displayed dose-dependent decreases in daily food intake and body weight, neither of these measures was influenced by any dose of DPN. In addition, DPN failed to modulate the anorexigenic effect of PPT when the two ER agonists were coadministered. Meal pattern analysis revealed that the anorexigenic effect of 75 microg PPT (a dose of PPT that produced a similar decrease in daily food intake as 4 microg EB) was mediated by a decrease in meal size, not meal number. Thus, PPT, like EB and endogenous estradiol, decreases food intake by selectively affecting the controls of meal size. The finding that acute administration of 75 microg PPT failed to induce a conditioned taste aversion suggests that the anorexigenic effect of this dose of PPT is not secondary to malaise. Taken together, our findings demonstrate that selective activation of ER alpha decreases food intake, body weight, and meal size in the ovariectomized rat.     

Cyclic estradiol treatment phasically potentiates endogenous cholecystokinin's satiating action in ovariectomized rats.

The influence of ovarian cycling and of exogenous estradiol on the cholecystokinin (CCK) satiety-signalling system was investigated in intact and ovariectomized Long-Evans rats, respectively. Intraperitoneal injection of 1 mg/kg devazepide, the most potent and selective CCK(A) receptor antagonist, increased test meal size during estrus, but not during diestrus, confirming the influence of hypothalamic-pituitary-gonadal function on CCK satiety in intact rats. Devazepide was then tested in ovariectomized rats that received chronic cyclic estradiol (2 microg estradiol benzoate on Tuesday and Wednesday each week) or oil treatment. Devazepide did not increase meal size in estradiol-treated rats on Tuesday, prior to estradiol treatment, compared to oil-treated rats, but did selectively increase meal size on Friday, late in the estradiol replacement cycle, compared to Tuesday, early in the cycle. These results suggest that a phasic potentiation of the endogenous CCK satiety-signalling system is part of the mechanism for the decrease in meal size in female rats during estrus.     

Peptides that regulate food intake: glucagon-like peptide 1-(7-36) amide acts at lateral and medial hypothalamic sites to suppress feeding in rats

Glucagon-like peptide 1-(7-36) amide (GLP-1) potently inhibits rat feeding behavior after central administration. Because third ventricular injection of GLP-1 appeared to be less effective than lateral ventricular injection, we have reexamined this issue. In addition, we attempted to identify brain regions other than the paraventricular nucleus of the hypothalamus that are sensitive toward GLP-1-induced feeding suppression. Finally, we examined the local role of endogenous GLP-1 by specific GLP-1 receptor blockade. After lateral ventricular injection, GLP-1 significantly inhibited food intake of 24-h-fasted rats in a dose-dependent fashion with a minimal effective dose of 1 microg. After third ventricular injection, GLP-1 (1 microg) was similarly effective in suppressing food intake, which extends previous findings. Intracerebral microinjections of GLP-1 significantly suppressed food intake in the lateral (LH), dorsomedial (DMH), and ventromedial hypothalamus (VMH), but not in the medial nucleus of the amygdala. The minimal effective dose of GLP-1 was 0.3 microg at LH sites and 1 microg at DMH or VMH sites. LH microinjections of exendin-(9-39) amide, a GLP-1 receptor antagonist, at 1 or 2.5 microg did not alter feeding behavior in 24-h-fasted rats. In satiated animals, however, a single LH injection of 1 microg exendin-(9-39) amide significantly augmented food intake, but only during the first 20 min (0.6 vs. 0.1 g). With three repeated injections of 2.5 microg exendin-(9-39) amide every 20 min, 1-h food intake was significantly increased by 300%. These data strongly support and extend the concept of GLP-1 as a physiological regulator of food intake in the hypothalamus.     

Influence of the subfornical organ on meal-associated drinking in rats

A lesion of the subfornical organ (SFO) may disrupt drinking after a meal of dry chow as it does drinking after intragastric administration of hypertonic saline. Food and water intakes of SFO-lesioned (SFOX) and sham-lesioned rats were measured during 90-min tests following various lengths of food deprivation. During the tests, all rats began eating before they began drinking. After 20-24 h of food deprivation, latency to begin drinking after eating had started was longer for SFOX than for sham-lesioned rats. Plasma osmolality was elevated by 2-3% in both lesion groups at 12 min, the latency for sham-lesioned rats to drink, but SFOX rats nevertheless continued eating and delayed drinking. Eating after shorter 4-h food deprivations and ad libitum feeding produced more variable drinking latencies and less consistent effects of SFO lesion. During 24 h of water deprivation, SFO lesion had no effect on the suppression of food intake and did not affect food or water intakes during the first 2 h of subsequent rehydration. These findings indicate that the SFO is involved in initiating water intake during eating and in determining drinking patterns and the amount of water ingested during a meal.     

The GLP-1 agonist exendin-4 reduces food intake in nonhuman primates through changes in meal size

Exendin-4 (Ex4), a long-acting glucagon-like peptide-1 (GLP-1) receptor agonist, has been shown to reduce food intake and suppress gastric emptying in rodents and humans. In this study we investigated the effects of peripheral administration of Ex4 on food intake and meal patterns in adult male rhesus macaques. Rhesus macaques (n = 4) that had been trained to lever press for food pellets were injected intramuscularly 15 min before the start of their 6-h daily feeding period. Ex4 was given at doses of 0.10, 0.32, 0.56, 1.0, and 3.0 microg/kg. Ex4 suppressed food intake in a dose-dependent manner, with the 3.0 microg/kg dose completely preventing feeding during the 6-h period and the 0.10 microg/kg dose suppressing intake by 17%. Doses of 0.32, 0.56, 1.0, and 3.0 microg/kg caused significant reductions in cumulative intake at all six hourly time points. Ex4 inhibited food intake through a specific effect on meal size. Meal size was significantly reduced in a dose-dependent manner with significant reductions at the 0.32 and 1.0 microg/kg doses (P < 0.05). Day 2 and 3 intakes returned to baseline levels with no compensation for Ex4-induced feeding suppression. Administration of doses of 0.32 and 0.56 microg/kg Ex4 over 5 consecutive days led to sustained reductions in intake with no evidence of compensation. Again, these reductions were due to specific effects on meal size. These results demonstrate that activation of GLP-1 pathways has potent effects on the controls of meal size and overall food intake in a nonhuman primate model.     

The naturally occurring C-17 fatty acid esters of estradiol are long-acting estrogens

C-17 fatty acid esters of estradiol are naturally occurring biosynthetic metabolites of estradiol. A representative component of this family of esters, estradiol-17-stearate, was studied in order to determine the estrogenic properties of these unusual hydrophobic steroids. Following the classical estrogen bioassay, a solution of this ester in oil was injected subcutaneously into immature rats once a day for 3 days. There was little effect on the uterus on the first day after the third injection. However, on subsequent days a large stimulation of uterine growth occurred. The course of this estrogenic effect was exactly opposite to that obtained with estradiol. In order to eliminate the possibility that this effect on the time course of estrogenic stimulation was caused by increased solubility of the hydrophobic esters in the carrier oil, the steroids were administered to adult ovariectomized animals in aqueous medium via a single intravenous injection. The uterotrophic response to estradiol was maximal at 12 h and was completely dissipated in 48-60 h. Estradiol-17-stearate produced a uterotrophic effect of twice the duration of estradiol. In the immature rat, aqueous intravenous injections of estradiol-17-stearate produced a greater uterotrophic effect than estradiol and this effect was still maximal 96 h later. In addition, this single injection of estradiol-17-stearate advanced the time of vaginal opening, a marker for puberty in the female rat. The mechanism of the prolonged estrogenic stimulation was investigated by studying the steroidal content of the uterus after injecting [3H]estradiol and [3H]estradiol-17 -stearate i.v. into immature rats. At 1 and 4 h there was significantly more radioactivity in the uteri of the [3H]estradiol treated animals. At later times (8 h and onwards) the total radioactivity in the uterus did not differ appreciably between the two groups. However at these later times, the amount of [3H]estradiol was far greater in the uteri of animals receiving [3H]estradiol-17-stearate. Consequently, the prolonged estrogenic effects of the endogenous C-17 fatty acid esters of estradiol are caused by the increased duration of the estrogenic signal. It is hypothesized that one of the roles of the fatty acid is to protect the steroid nucleus from metabolism and thereby prolong the life of the parent C18 steroid. Thus, the results of these experiments are consistent with the family of endogenous alkyl esters of estradiol having a physiological role as long-acting estrogens.     

Effects of cholecystokinin octapeptide (CCK-8) on food intake in adult and aged rats under different feeding conditions

The effects of CCK on food intake were investigated under fixed feeding conditions in comparison to a test meal taken after 16 h of food deprivation. The experiments were performed on young adult rats (8 weeks old) as well on aged rats (23 months old). Intraperitoneal CCK-8 (8 and 40 μg/kg) significantly reduced the size of a test meal following 16-h food deprivation. This effect was independent of the age of the rats. However, under fixed feeding conditions neither of the doses used in this study reduced food intake in the young adult rats, whereas the highest dose of 40 μg/kg did so in the aged rats. These results suggest that the hypophagic effect of exogenous CCK-8 depends on experimental conditions, food intake being reduced after a period of food deprivation but not under a fixed feeding regimen in adult animals. Furthermore, the data suggest that age is a factor contributing to the complex behavioral actions of CCK, because only old animals were more susceptible to an anorectic action of CCK under the fixed feeding schedule. An explanation may lie in an interaction of other known behavioral effects of CCK (e.g., anxiogenic, mnemonic action) with its effects under the different feeding schedules.     

Reduced feeding during water deprivation depends on hydration of the gut

Removal of drinking water at the start of the dark period reduced food intake in freely feeding rats within 45 min. Both first and later meals were smaller during 7.5 h of water deprivation, but meal frequency did not change. Ingestion of a normal-sized meal (3 g) rapidly increased plasma tonicity when drinking water was withheld, but intravenous infusions of hypertonic NaCl causing similar increases in plasma tonicity did not reduce feeding. Feeding during 6 h of water deprivation was restored by slowly infusing the volume of water normally drunk into the stomach, jejunum, or cecum, but not in the vena cava or hepatic portal vein. The infusions did not alter water or electrolyte excretion or affect food intake in rats allowed to drink. We conclude that the inhibition of feeding seen during water deprivation is mediated by a sensor that is located in the gastrointestinal tract or perhaps in the mesenteric veins draining the gut, but not the hepatic portal vein or the liver. In the absence of drinking water, signals from this sensor provoke the early termination of a meal.     

Effect of naloxone on pancreatic and gastric endocrine function in response to carbohydrate and fat-rich test meals

The present study was designed to determine the effect of naloxone, a specific opiate receptor antagonist, on postprandial levels of insulin, glucagon, pancreatic polypeptide (PP), somatostatin-like immunoreactivity (SLI) and gastrin in response to carbohydrate and fat-rich test meals in a group of 6 healthy volunteers. The addition of naloxone to a meal consisting of 50 g sucrose dissolved in 200 ml water augmented the rise of plasma insulin levels significantly during the first 30 min after its ingestion and reduced the decrease of plasma glucagon. During the ingestion of a fat-rich meal in form of 200 ml cream naloxone reduced the rise in plasma insulin and pancreatic polypeptide and elevated glucagon levels during the last 30 min of the experimental period. When sucrose was dissolved in 200 ml cream the addition of naloxone augmented the postprandial rise of insulin levels between 15 and 60 min after ingestion of the meal and elicited an increase of plasma SLI and PP levels throughout the entire experimental period which indicates that post-prandial levels of insulin, glucagon, PP and SLI are modulated via endogenous opiate receptors during the ingestion of carbohydrate and fat test meals and that this effect depends on the composition of the ingested nutrients. These data raise the possibility that endogenous opiates participate in the regulation of postprandial insulin, glucagon, somatostatin and pancreatic polypeptide release not only in certain disease states as demonstrated recently for insulin secretion in type II diabetes mellitus but endogenous opiates may also be of importance under physiological conditions.     

Can one unrestricted meal buffer the effects of previous pre-meal intervals on the feeding behaviour of sheep?

Varying the time since the last meal is one means of manipulating feeding motivation. In order to use this method effectively it is necessary to know whether and the extent to which effects of one pre-meal interval are carried over to affect the behaviour during the following meals. Pre-meal interval (PMI) is defined here for practical purposes, for short meals, as the time between the start of two successive meals. The possibility that one unrestricted meal might buffer the effects of an 8h as opposed to a 4h PMI on aspects of feeding behaviour was studied with eight Scottish Blackface sheep. They were fed on a regime in which they were given access to food until they finished their meal and lay down (this always occurred within 60min) at which time the remaining food was withdrawn. Feeding behaviour was recorded during the meal after these 4 and 8h intervals, as well as during the following meal 4h later.At a meal after a PMI of 8h, compared to 4h, sheep had a higher intake per meal (mean+/-S.T.D. for 8 and 4h PMI, respectively: 604.4+/-78.8 and 430.1+/-100.9g; P<0.001), a longer meal duration (27.1+/-7.5 and 21.8+/-8.1min; P<0.001), and a tendency for a higher intake rate (23.8+/-6.2 and 21.9+/-8.2g/min; P=0.11). During the following meal 4h later these differences were smaller, but intake per meal still tended to be higher (430.8+/-81.5 and 338.5+/-45.6g; P<0.06) for sheep who had previously had the 8h PMI. Meal duration (21.9+/-7.2 and 20.6+/-7.08min; P=0.28) and intake rate (21.2+/-6.1 and 18.7+/-7.2g/min; P=0.13) were no longer different.A single meal after the different PMIs reduced differences in all three aspects of feeding behaviour observed during the subsequent meal, 4h later, but differences in intake per meal were still apparent. It is suggested that an additional meal may overcome the carry-over effect.     

Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation

Feeding promotes protein accretion in skeletal muscle through a stimulation of the mRNA translation initiation phase of protein synthesis either secondarily to nutrient-induced rises in insulin or owing to direct effects of nutrients themselves. The present set of experiments establishes the effects of meal feeding on potential signal transduction pathways that may be important in accelerating mRNA translation initiation. Gastrocnemius muscle from male Sprague-Dawley rats trained to consume a meal consisting of rat chow was sampled before, during, and after the meal. Meal feeding enhanced the assembly of the active eIF4G.eIF4E complex, which returned to basal levels within 3 h of removal of food. The increased assembly of the active eIF4G.eIF4E complex was associated with a marked 10-fold rise in phosphorylation of eIF4G(Ser(1108)) and a decreased assembly of inactive 4E-BP1.eIF4E complex. The reduced assembly of 4E-BP1.eIF4E complex was associated with a 75-fold increase in phosphorylation of 4E-BP1 in the gamma-form during feeding. Phosphorylation of S6K1 on Ser(789) was increased by meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of mammalian target of rapamycin (mTOR) on Ser(2448) or Ser(2481), an upstream kinase responsible for phosphorylating both S6K1 and 4E-BP1, was increased at all times during meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of PKB, an upstream kinase responsible for phosphorylating mTOR, was elevated only after 0.5 h of meal feeding for Thr(308), whereas phosphorylation Ser(473) was significantly elevated at only 0.5 and 1 h after initiation of feeding. We conclude from these studies that meal feeding stimulates two signal pathways in skeletal muscle that lead to elevated eIF4G.eIF4E complex assembly through increased phosphorylation of eIF4G and decreased association of 4E-BP1 with eIF4E.