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The recently discovered orexigenic peptide ghrelin is produced primarily by the stomach and circulates in blood at levels that increase during prolonged fasting in rats. When administered to rodents at supraphysiological doses, ghrelin activates hypothalamic neuropeptide Y/agouti gene-related protein neurons and increases food intake and body weight. These findings suggest that ghrelin may participate in meal initiation. As a first step to investigate this hypothesis, we sought to determine whether circulating ghrelin levels are elevated before the consumption of individual meals in humans. Ghrelin, insulin, and leptin were measured by radioimmunoassay in plasma samples drawn 38 times throughout a 24-h period in 10 healthy subjects provided meals on a fixed schedule. Plasma ghrelin levels increased nearly twofold immediately before each meal and fell to trough levels within 1 h after eating, a pattern reciprocal to that of insulin. Intermeal ghrelin levels displayed a diurnal rhythm that was exactly in phase with that of leptin, with both hormones rising throughout the day to a zenith at 0100, then falling overnight to a nadir at 0900. Ghrelin levels sampled during the troughs before and after breakfast correlated strongly with 24-h integrated area under the curve values (r = 0.873 and 0.954, respectively), suggesting that these convenient, single measurements might serve as surrogates for 24-h profiles to estimate overall ghrelin levels. Circulating ghrelin also correlated positively with age (r = 0.701). The clear preprandial rise and postprandial fall in plasma ghrelin levels support the hypothesis that ghrelin plays a physiological role in meal initiation in humans. [ABSTRACT FROM AUTHOR]

Objective: The goal of this study was to determine whether differential induction of skeletal muscle uncoupling protein 3 (UCP3) contributes to the development of diet-induced obesity (DIO) or resistance to the development of obesity (DR) when rats are placed on a moderate fat (31%) high energy (HE) diet., Research Methods and Procedures: Gastrocnemius muscle was obtained from Sprague-Dawley rats that were identified as DIO-prone (n = 5) or DR (n = 5) on the basis of urinary norepinephrine excretion while consuming a chow diet. Muscle was also obtained from animals in the top tertile of weight gain (DIOHE, n = 5) and the bottom tertile of weight gain (DRHE, n = 5) after 2 weeks on the HE diet. UCP3 and actin mRNA levels were measured in all muscle samples by Northern analysis. To distinguish the effect of dietary energy content from the effect of obesity itself, we studied additional DIO and DR animals that had been returned to a chow diet for 10 weeks after consuming a HE diet for 10 weeks., Results: The muscle UCP3/actin mRNA ratio in animals that resisted the development of obesity during 2 weeks on the HE diet was 3-fold higher than in the other groups (DRHE = 3.24 +/- 0.83, DIOHE = 0.91 +/- 0.20, DIO-prone = 0.72 +/- 0.15, DR = 0.63 +/- 0.15; p = 0.002). However, there was no difference in muscle UCP3/actin mRNA ratios between DIO animals and DR animals that had been fed the HE diet for 10 weeks and then returned to either an ad libitum chow diet for 10 weeks (DIO = 13.8 +/- 3.53, DR = 11.1 +/- 3.43, p = NS) or to a restricted chow diet for 10 weeks (DIO = 11.0 +/- 2.85, DR = 10.6 +/- 2.20, p = NS) despite significantly greater body weight of the DIO animals., Discussion: DR animals may initially resist weight gain when placed on a HE diet through a greater induction of muscle UCP3. This induction is transient and is related more closely to dietary fat content than to body fat stores. DIO animals show no initial induction of muscle UCP3, which may contribute to their increased metabolic efficiency soon after exposure to a HE diet.

To determine whether leptin alone accounts for the satiety activity secreted by native adipose tissue, we prepared culture media conditioned by microdissected adipose tissue from overfed Long-Evans rats, fa/fa rats, or db/db mice (media A, B, and C, respectively). Medium A significantly suppressed food intake following intracerebroventricular delivery to Long-Evans rats (2-h chow intake = 68 +/- 5% of baseline, P < 0.001). Media B and C significantly suppressed food intake following intraperitoneal delivery to ob/ob mice (24-h chow intake = 56 +/- 7% of baseline for medium B, P = 0. 001; 4-day chow intake = 78 +/- 3% of baseline for medium C, P = 0. 004). Using a leptin receptor-based bioassay, we determined that the leptin concentration of medium C was 392 +/- 18 ng/ml. This concentration was 20-fold lower than the concentration of recombinant murine leptin required to produce a similar degree of feeding suppression following 5 days of administration to ob/ob mice. Neither medium conditioned by adipose tissue from ob/ob mice nor medium conditioned by adipose tissue from fa/fa rats and subsequently immunodepleted of leptin had significant satiety activity. We conclude that leptin is necessary but not sufficient to account for the satiety activity of native adipose tissue, perhaps due to the production by adipocytes of a cofactor that augments the ability of leptin to suppress feeding.

The timing of puberty onset in mammals is tightly coupled to the animal's nutritional and metabolic state. We conducted two experiments to test the hypothesis that leptin acts as a metabolic signal for the onset of puberty. In the first experiment, we administered leptin (6.3 micrograms/g twice daily) to a group of normal prepubertal female rats and compared their rate of sexual maturation to that of two control groups. The group of leptin-treated animals and one group of control animals were allowed to eat ad lib, while the other group of control animals was pair-fed to the leptin-treated group. Food intake in the leptin-treated group was reduced to approximately 80% of the ad lib-fed control group, resulting in retarded growth in both leptin-treated and pair-fed animals. All measured indices of pubertal maturation-age at vaginal opening, age at first estrus, ovarian weight, ovulatory index (corpora lutea/ovarian section), uterine weight, and uterine cross-sectional area-were significantly delayed in the pair-fed group but not different between the leptin-treated group and ad lib-fed controls. The second experiment was similar to the first, except that both the leptin-treated group and the pair-fed group were fed at 70% of the ad lib-fed controls. Under these conditions, leptin only partially reversed the delay in sexual maturation, as reflected by the age at vaginal opening and first estrus. These results suggest that leptin is not the primary signal that initiates the onset of puberty but that instead, it acts in a permissive fashion, as a metabolic gate, to allow pubertal maturation to proceed-if and when metabolic resources are deemed adequate; moreover, these observations suggest that other metabolic factors, besides leptin, influence the timing of puberty onset under conditions of more severe dietary stress.

The view that the adipocyte acts only as a passive storage site for energy in the form of triglyceride has been rendered obsolete by the discovery that adipocytes secrete a variety of metabolically active molecules. These molecules include free fatty acids, which decrease the rate of glucose oxidation by peripheral tissues; adipsin and other complement factors involved in host defense; tumor necrosis factor alpha, which may be an important determinant of insulin sensitivity; and angiotensinogen, which appears to promote terminal differentiation of preadipose to adipose cells. Leptin, a 167 amino acid polypeptide encoded by the obese gene, is a recently described adipocyte secretory product that communicates the status of the body's energy reserve to the central nervous system, apparently for the purpose of regulating body composition. Plasma leptin levels are exponentially related to total adipose mass. Daily injection of leptin into ob/ob mice leads to decreased food consumption and increased energy expenditure, both of which result in loss of adipose mass. Leptin-treated animals also have lower circulating insulin and glucose levels than pair fed controls. Finally, leptin corrects the infertility of ob/ob mice by restoring gonadotropin secretion to normal. These observations indicate that the adipocyte plays a key role in energy balance, insulin action, host defense, and reproduction, and suggest new approaches for understanding several important human diseases.

Physiological investigation has demonstrated that the central nervous system monitors body composition and adjusts energy intake and expenditure to stabilize total adipose tissue mass. Genetic variations in the signalling molecules involved in this regulatory system account for the heritable component of body fat content. The application of molecular techniques to rodent models of Mendelian obesity has resulted in the characterization of five loci at which mutations produce an abnormal accumulation of body fat. The genes at these loci include agouti, which encodes a molecule that antagonizes the binding of alpha melanocyte-stimulating hormone to its receptor; fat, which encodes carboxypeptidase E; tubby, which encodes a putative phosphodiesterase; obese, which encodes a circulating satiety protein; and diabetes, which encodes the receptor for the obese gene product. A more detailed understanding of the functional interrelationships of these genes should lead to important new insights into the causes and potential therapies for human obesity.

Stability of body composition requires that energy intake equals energy expenditure when integrated over prolonged periods. As recent human studies have failed to demonstrate active changes in energy expenditure with changes in body composition, it is likely that energy intake is continually adjusted to preserve a constant total adipose tissue mass. If adipose tissue mass is regulated directly, then there must be some input reflecting this quantity to the central nervous system for the purpose of making corrective changes in appetite when total body fat content fluctuates. The nature of this input has been examined in a variety of animal experiments involving induced weight change, lipectomy, plasma transfer from obese or satiated animals to hungry animals, and parabiosis between obese and lean animals. The bulk of evidence suggests that the plasma level of one or more currently unidentified stable circulating molecules increases in proportion to total body fat content and augments the effect of meal-related satiety signals in the central nervous system. The implications of this adipose tissue-related satiety factor for the pathogenesis of obesity, and the possible nature of the factor are discussed.

This discussion was selected from the weekly Grand Rounds in the Department of Medicine, University of Washington School of Medicine, Seattle. Taken from a transcription, it has been edited by Drs Paul G. Ramsey, Associate Professor of Medicine, and Philip J. Fialkow, Professor and Chair of the Department of Medicine.

A technique based on a completely metabolizable liquid diet was developed rapidly to achieve weight stability (+/- 50 g/day) in experimental subjects. Weight-stabilizing caloric intake was then used as an estimate of total daily energy expenditure (24 h EE) in a study of 10 ambulatory obese subjects before and after weight loss. In five control subjects 24 h EE fell by 928 +/- 121 kcal/day (3885 +/- 507 kJ/day) or 29 +/- 3 percent after a 22.6 +/- 1.2 percent weight loss achieved on a three month 700 kcal/day (2930 kJ/day) diet. To determine how much of this decrease was due to the reduced thermic effect of exercise (TEE) at the lower body weight, the remaining five subjects underwent progressive exogenous weight replacement during dieting to compensate exactly for their 21.0 +/- 5.2 percent weight loss. In the latter group 24 h EE fell by only 392 +/- 192 kcal/day (1641 +/- 804 kJ/day) or 12 +/- 5 percent (P less than 0.001). These data, along with indirect calorimetric measurements of resting metabolic rate (RMR), permitted the decrease in 24 h EE with weight loss to be divided into its component parts. Decreases in RMR, TEE and the thermic effect of food accounted for 30 percent, 52 percent and 18 percent respectively of the fall in 24 h EE. A reduction in TEE appears to make a greater contribution to the enhanced energy efficiency of ambulatory reduced-obese subjects than has been suggested by studies based on methods requiring greater subject confinement.

The extent to which the resting and nonresting components of 24-hour energy expenditure decrease after weight reduction has not been prospectively assessed in ambulatory, weight-stable, reduced-obese humans. Accordingly, 24-hour energy expenditure was estimated as the weight-stabilizing (+/- 50 g/d) daily caloric intake of a defined liquid diet in a cross-sectional study of ten reduced-obese subjects after a 23.2% +/- 9.4% weight loss and 18 obese subjects at baseline weight. A regression analysis demonstrated an 18% decrease in the mean daily energy requirement of the reduced-obese subjects compared with that of subjects of the same relative body weight who had never dieted. Strong linear relationships were noted between estimated 24-hour energy expenditure and fat-free mass (FFM), and between resting metabolic rate (RMR) and FFM in the subjects at baseline weight. In six reduced-obese men, the 24-hour energy expenditure was only 75.7% +/- 5.6% of the value predicted by regression analysis for the decreased FFM. In these six subjects the RMR was 97.4% +/- 7.5% of that predicted for the decreased FFM, suggesting that essentially all the energy savings relative to FFM in the reduced-obese state occurred in nonresting energy expenditure. In a subsequent group of seven subjects studied longitudinally before and after a 21.5% +/- 2.3% weight loss, the decrease in nonresting energy expenditure accounted for 582 +/- 276 kcal/d or 71% of the decrease in estimated 24-hour energy expenditure. These data suggest a decrease in the nonresting energy expenditure of ambulatory reduced-obese individuals, which is greater than previously appreciated.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatalities accompanying the use of very low calorie diets have been attributed to cardiac arrhythmias, which may have been associated with myocardial catabolism and hypokalemia. Delayed activation of damaged regions of myocardium may result in low amplitude potentials late in the QRS complex which have been associated with sustained ventricular tachycardia. We used the techniques of routine electrocardiography, signal averaging of the surface electrocardiogram and ambulatory electrocardiographic (Holter) monitoring to evaluate a group of 11 obese men (116 +/- 14 kg) who consumed a potassium-supplemented 660 kcal/day (2763 kJ/day) milk-based liquid diet for 95 +/- 14 days. Electrocardiographic measurements were made before beginning the diet, during the final week on the diet, and after refeeding. Corrected QT interval, QRS voltage and duration, absence of signal-averaged late potentials and ventricular ectopy during 24 h Holter monitoring during the diet and refeeding did not differ from baseline determinations at any point in the obese subjects. In this study, which involved only 11 subjects, neither standard electrocardiographic techniques nor a newer signal-averaging technique revealed any electrocardiographic changes from the diet. A very large population would have to be studied to evaluate the usefulness of these techniques in predicting myocardial damage serious enough to cause arrhythmias. Until such studies are available, the safety of such diets remains unknown.

We have reported that glucagon administered to perifused rat hepatocytes as a series of pulses at 15-min intervals is a more effective stimulus for hepatocyte glucose production (HGP) than is continuous glucagon infusion. To test whether the efficiency of HGP depends upon the frequency of pulsatile glucagon delivery, we administered glucagon to perifused rat hepatocytes as a series of pulses of fixed amplitude [922 +/- 30 (+/- SE) pg/ml] at eight separate pulse intervals ranging from 3-45 min. Compared to continuous infusion of the same total amount of hormone, pulsatile glucagon administration clearly enhanced HGP in a frequency-dependent fashion. At pulse intervals between 10 and 20 min, pulsatile HGP exceeded continuous HGP by a factor of 1.5-2. This range of optimal intervals compared favorably with the glucagon secretory period of 10 min observed in nonhuman primates and that of 13-20 min observed in humans. We noted a desensitization of the hepatocyte response to glucagon that was directly proportional to the log of the time-averaged hormone concentration. Since the magnitude of the desensitization elicited by pulsatile glucagon delivery exceeded the desensitization elicited by continuous hormone delivery regardless of pulse frequency, differential desensitization could not explain the frequency dependency of the pulse enhancement effect. A mathematical simulation of our data demonstrated that the asymmetry of the HGP waveform elicited by a brief glucagon pulse could account for the observed frequency dependency of HGP. Pulse to pulse summation and the desensitization phenomenon modulated both the magnitude of the pulse enhancement effect and the frequency range over which the effect was manifest. We conclude that the enhancement of HGP by glucagon pulses is a frequency-dependent phenomenon and that the physiological glucagon secretory period optimizes HGP.

Reduced energy expenditure after weight loss contributes to the difficulty which obese individuals face in maintaining a lower body weight. To evaluate the contribution of decreased energy cost of physical activity at a lower body weight to diminished total energy expenditure, two groups of unconfined obese men were studied before and after a 22 percent weight loss. A control group received a 700-cal diet alone whereas the experimental group, while consuming an identical diet, wore a vest into which weights were inserted twice weekly to exactly compensate for body weight loss. In the control group the total daily caloric requirement for weight maintenance fell by 916 +/- 121 kcal/day (3834 +/- 507 kJ/day) in contrast to only a 404 +/- 192 kcal/day (1691 +/- 804 kJ/day) fall in the experimental group at full exogenous weight replacement (P less than 0.001). Body composition, maximal exercise performance, and resting metabolic rate were unaffected by weight replacement. Activity levels decreased at the same rate in both subject groups with progressive weight loss. In conclusion, loss of body weight reduces the energy cost of physical activity sufficiently to account for more than half of the major fall in energy expenditure noted in free-living men after weight loss.