Françoise J. Smith

Targeted disruption of the melanocortin-4 receptor results in obesity in mice

The melanocortin-4 receptor (MC4-R) is a G protein-coupled, seven-transmembrane receptor expressed in the brain. Inactivation of this receptor by gene targeting results in mice that develop a maturity onset obesity syndrome associated with hyperphagia, hyperinsulinemia, and hyperglycemia. This syndrome recapitulates several of the characteristic features of the agouti obesity syndrome, which results from ectopic expression of agouti protein, a pigmentation factor normally expressed in the skin. Our data identify a novel signaling pathway in the mouse for body weight regulation and support a model in which the primary mechanism by which agouti induces obesity is chronic antagonism of the MC4-R.

Human eating : evidence for a physiological basis using a modified paradigm

The aim of these studies was to determine if meal requests and changes in hunger ratings in humans were related to spontaneous changes in blood glucose concentration. In our first study, 18 healthy subjects were acutely isolated from food ant time cues. Blood glucose was continuously monitored online and visual analog ratings of hunger were obtained following an overnight fast. Spoken meal requests, if they occurred, were also recorded. In 83% of the subjects, both the perception and behavioral expression of hunger, as assessed by changes in hunger ratings and meal requests, were preceded by, and correlated with, brief, transient declines in blood glucose (nadir: -10% at 27 min). The pattern, magnitude and time course of these declines was similar to those observed in rats. This significant association, between increased expression of hunger and declines in blood glucose, is being tested in a second, ongoing study using acute insulin infusions to mimic spontaneous transient declines in blood glucose. Each subject was studied twice: either insulin or saline was infused while hunger ratings were obtained. Preliminary results in five subjects indicate that hunger ratings increased after insulin-induced transient declines in blood glucose. No change in hunger ratings occurred when blood glucose concentration was stable. These results suggest that this temporal pattern of blood glucose reflects an antecedent physiological event or provides a signal related to the expression of hunger in humans. Further understanding of human eating may result from investigation of the complex interaction of physiological and other factors in an experimental setting that allows the expression the behavior under study.

Chronic administration of OB protein decreases food intake by selectively reducing meal size in female rats.

The potent hypophagic effect of OB protein (OB) is well established, but the mechanism of this effect is largely unknown. We investigated the effects of chronic administration of a novel modified recombinant human OB (Mod-OB) with a prolonged half-life (>48 h) on ad libitum food intake, spontaneous meal patterns, and body weight in 24 adult, male Sprague-Dawley rats (body weight at study onset: 292 g). Single daily subcutaneous injections of Mod-OB (4 mg/kg daily) for 8 consecutive days significantly reduced ad libitum food intake compared with vehicle injections from injection day 3 through postinjection day 3. Mod-OB-injected rats ate between 4.5 and 7.1 g (or 13-20%) per day less than controls, with the reduction primarily occurring during the dark period. Body weight gain was significantly decreased in response to Mod-OB from injection day 8 until postinjection day 4, with a maximum difference of 24 g on postinjection day 3. The reduction of food intake by Mod-OB was mainly due to a 21-34% decrease in nocturnal spontaneous meal size. There was no significant effect of Mod-OB on nocturnal meal frequency or duration. Mod-OB also did not reliably affect the size, duration, or frequency of diurnal meals. Mod-OB-injected rats displayed no compensatory hyperphagia after the injection period. These results indicate that chronically administered OB selectively affects the mechanisms controlling meal size in male rats.

Overview: neurobiology of OB protein (leptin).

The rapid elucidation of the properties, target tissues and actions of OB protein, which is the product of the ob gene, has invigorated and energized obesity research as no other finding in this field has in the last 35 years. The circulating concentrations of OB protein are proportional to adiposity and increase with increasing levels of body fat (Considine et al. 1996). The OB protein pathway is the long-sought hormonal signal pathway from adipose tissue to the brain that plays a critical role in the regulation of energy balance (Kennedy, 1953; Campfield etal. 1996a, 1997~). OB protein is a 16 kDa polypeptide hormone that is secreted from adipose tissue, circulates in the blood, bound to a family of binding proteins, enters the brain, binds to its receptor in hypothalamic nuclei and other brain areas and acts on central neural networks. Available evidence suggests that OB protein appears to play a major role in the control of body fat stores through co-ordinated regulation of feeding behaviour, metabolism, neuroendocrine responses, autonomic nervous system and body energy balance in rodents, primates and man (Campfield et al. 1996a, 1997~). The mechanisms in the brain responsible for determining the level at which body fat content is regulated in man and other animals are not completely understood. A similar lack of knowledge exists for the mechanisms regulating the neuroendocrine rhythms supporting the adaptation to starvation and reproductive function in man and other animals. Elucidation of the OB protein pathway within the brain has begun to provide important insights into these, and possibly other, mechanisms. If OB protein proves to be a useful tool to illuminate the mechanisms controlling body fat content and its corresponding decision rules or algorithms, it may provide the basis for a clearer understanding of the regulation of body fat content and energy balance (Campfield et al. 1996a, 1997~). When these mechanisms are understood at the molecular level, they should provide new targets for therapeutic interventions that will reduce and maintain body fat at reduced levels and, therefore, increase metabolic fitness, reduce risk factors and promote improved health of obese individuals. It is this hope that creates much of the excitement that greets each research advance in the understanding of the OB protein pathway (Campfield et al. 1996a, 1997a). Since the cloning of the ob gene in December 1994 (Zhang et al. 1994), research has progressed along four parallel paths: (1) regulation of ob gene expression in adipose tissue in mice, rats and man: (2) characterization of the biological actions and definition of the elements of the OB protein pathway in lean and obese mice and rats; (3) studies of the biology of OB protein in lean and obese human subjects; (4) studies of the brain structures and mechanisms through which OB protein acts. The key elements of the OB protein pathway are shown in Figure 1, including a transport system for OB protein to enter the brain, OB protein receptors in hypothalamic nuclei, and neural and neuroendocrine outputs to peripheral tissues (Campfield et al. 1996a, 1997~). In the present review we will discuss the available knowledge about the OB protein pathway within the brain. This understanding is based on in vitro experiments as well as studies conducted in laboratory animals and human subjects. This research has revealed that the OB protein, by acting on diverse brain structures and mechanisms, regulates ingestive behaviour, metabolism, neuroendocrine rhythms and controls body energy balance. These brain structures and mechanisms form the central OB protein pathway. The role of OB protein, the concept of reduced brain sensitivity to OB protein in obesity, and possible therapeutic approaches based on OB protein will also be presented. Finally, the interaction of OB protein with other brain mechanisms is summarized. The role of OB protein in obesity and the future of neurobiology of OB protein will also be discussed.

Regional localization of specific [125I]leptin binding sites in rat forebrain

Specific [125I]leptin receptor binding sites have been identified in choroid plexus (CP), but have eluded regional localization within the brain parenchyma. To optimize specific [125I]leptin binding in brain loci, we ran experiments varying the pH of incubation buffers. We found that specific [125I]leptin binding in CP was strikingly pH dependent with the most acidic buffer, pH 5.5, resulting in a greater than 100% increase over the amount of specific binding measured at pH 7.5. While low pH permitted detection of specific binding in parenchymal loci, clear pH dependency was only observed in the CP. In the caudate putamen (CauP), a locus with low specific binding, values for specific binding did not differ significantly across the range of pH conditions tested. Using incubation buffers at pH 6.0 in subsequent binding experiments, we localized specific [125I]leptin binding in several brain loci including thalamus and hypothalamus. In CP and thalamus, where the range of OD permitted analysis of binding parameters, [125I]leptin binding was saturable with increasing concentrations of unlabelled leptin. In all loci, specific [125I]leptin binding was insensitive to competition by high concentrations of other unlabelled compounds. Our results varying pH conditions of the incubation buffer suggest leptin receptors may be divided into subclassifications based on pH sensitivity of the specific binding. Furthermore, our results suggest that although densities are low, high affinity leptin receptors are present in neural loci implicated in food intake and energy balance, and are more widespread in the forebrain than previously determined.

Brain administration of OB protein (leptin) inhibits neuropeptide-Y-induced feeding in ob/ob mice

OB protein (or leptin) administration causes a long-lasting reduction in food intake and body weight in obese ob/ob mice. Neuropeptide Y, a stimulator of feeding, has been proposed to be a major mediator of the biological actions of OB protein. To test this hypothesis, the interaction of brain administration of exogenous OB protein and NPY on the feeding behavior of ob/ob mice was examined. Human OB protein, in a dose-dependent manner, partially or completely blocked feeding induced by exogenous NPY. These results demonstrate that OB protein can functionally antagonize and dominate the actions of exogenous NPY on feeding.

Blood Glucose Patterns and the Control of Feeding Behavior: A New Framework for the Control of Meal Initiation

A new framework for understanding the control of feeding behavior, with special emphasis on the evolution of hunger, the initiation of feeding, and its dependence on patterns of blood glucose, is the subject of this chapter. A perspective on the current status and future directions of this search for a more complete understanding of the regulation of feeding behavior in laboratory rats and humans is presented, including theoretical and experimental components. Experimental evidence supports the hypothesis that spontaneous, self-resolving transient declines in blood glucose precede and signal meal initiation in nondeprived, free-feeding rats and time-isolated humans. This signal precedes food-seeking behavior and the initiation of a meal but does not predict the size of the meal or the timing of meal termination. The precise antecedent conditions required, in terms of the shape of the transient declines in blood glucose, for meal initiation or meal requests have been defined. This is followed by a statement and overview of a signal detection and pattern recognition theory of the control of meal initiation. The current working hypothesis that transient declines in blood glucose are endogenous metabolic patterns that are represented in the central nervous system is then presented. These patterns are detected and recognized by the central nervous system and are mapped into meal initiation in rats and are correlated with meal requests in humans. The distinguishing feature of the theory is that it is the temporal pattern, shape, or waveform of blood glucose dynamics – rather than the glucose molecule, or the absolute decrease in blood glucose, or blood glucose concentration, or glucose utilization – that is detected and contains critical information that is extracted by the central nervous system to control meal initiation. Then, the experimental studies on meal initiation and its dependence on patterns of blood glucose in humans are reviewed. An association between transient declines in blood glucose concentration and meal requests and changes in hunger ratings in human subjects isolated from food and time cues has been demonstrated. This association was observed following both spontaneous and insulin-induced transient declines in blood glucose. These results support and strengthen the conclusion that the transient decline in blood glucose represents a temporal pattern that reflects an antecedent physiological event or provides a signal related to the expression of hunger in humans. Finally, the implications for the understanding of the control of feeding behavior and the regulation of energy balance are discussed.