predicted that the POMC gene should be expressed in ARC neurons that do not contain Agrp mRNA. To determine if NPY mRNA is coexpressed with either Agrp or POMC mRNA in ARC neurons of fed and fasted mice and rats, we colocalized nature neuroscience • volume 1 no 4 • Neuropeptide Y (NPY) stimulates food intake and promotes weight gain, whereas melanocortins have the opposite effect. Yet both peptides are synthesized in the arcuate nucleus, a hypothalamic area involved in energy homeostasis. We report here that mRNA encoding NPY and the melanocortin precursor, proopiomelanocortin (POMC) are expressed in adjacent, but distinct, subpopulations of arcuate nucleus neurons. Moreover, these NPY neurons coexpress mRNA encoding Agouti-related protein (Agrp), an endogenous melanocortin receptor antagonist, and fasting increases the expression of both of these mRNA species. Our findings suggest that hypothalamic NPY/Agrp neurons constitute a unique cell type that is activated by fasting to stimulate food intake via a simultaneous increase of NPY and decrease of melanocortin.The ability to recover weight lost during periods of limited access to food is important for survival. Accordingly, a highly integrated central nervous system response to starvation and weight loss has evolved that includes changes in the activity of discrete hypothalamic pathways involved in energy homeostasis 1,2 . Components of this response include the activation of hypothalamic neurons that contain NPY (which stimulates food intake) coupled with reduced signaling by neurons that contain melanocortins (which inhibit food intake). Melanocortin-containing cell bodies in the forebrain have been detected only in the hypothalamic arcuate nucleus (ARC), the same nucleus that contains a population of NPY neurons that are implicated as critical in energy homeostasis 3 . Similarly, hypothalamic expression of Agrp 4,5 (also known as Agouti-related transcript, ART), a newly discovered antagonist of melanocortin receptors, has also been found only in neurons of the ARC.These anatomical considerations, as well as the ability of NPY and Agrp to promote increased food intake and weight gain 1,4 , led us to propose that these two gene products are expressed by the same ARC neurons, as proposed previously 5 . We hypothesized further that if NPY and Agrp are coexpressed by ARC neurons, their expression should be regulated in parallel during fasting. In contrast, because melanocortins reduce food intake 6 , we Fig. 1. Fluorescent in situ hybridization of mRNA encoding Agrp in the ARC of mice fed ad libitum (a) or fasted (b). Adult male mice (25 g) were fasted for 48 h. Brains were removed after decapitation, frozen on dry ice and sectioned at 14 µm. In situ hybridization used antisense Agrp cRNA probe. Sections were viewed with a Zeis Axioplan fluorescence microscope and quantified using MCID M2 imaging software (Imaging Research, Ontario, Canada). The expression of Agrp mRNA increases with fasting. All animals were treated in accordance with University of Washington gui...

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Hypothalamic melanocortins are among several neuropeptides strongly implicated in the control of food intake. Agonists for melanocortin 4 (MC-4) receptors such as -melanocyte-stimulating hormone ( -MSH), a product of proopiomelanocortin (POMC), reduce food intake, whereas hypothalamic agouti-related protein (AgRP) is a MC-4 receptor antagonist that increases food intake. To investigate whether reduced melanocortin signaling contributes to hyperphagia induced by uncontrolled diabetes, male Sprague-Dawley rats were studied 7 days after administration of streptozotocin (STZ) or vehicle. In addition, we wished to determine the e ffect of diabetes on muscle uncoupling protein 3 (UCP-3), a potential regulator of muscle energy metabolism. STZ diabetic rats were markedly hyperglycemic (31.3 ± 1.0 mmol/l; P < 0.005) compared with nondiabetic controls (9.3 ± 0.2 mmol/l). Insulin treatment partially corrected the hyperglycemia (18.8 ± 2.5 mol/l; P < 0.005). Plasma leptin was markedly reduced in STZ diabetic rats (0.4 ± 0.1 ng/ml; P < 0.005) compared with controls ( 3 . 0 ± 0.4 ng/ml), an effect that was also partially reversed by insulin treatment (1.8 ± 0.3 n g / m l ) . Untreated diabetic rats were hyperphagic, consuming 40% more food (48 ± 1 g/day; P < 0.005) than controls (34 ± 1 g/day). Hyperphagia was prevented by insulin treatment (32 ± 2 g/day). In untreated diabetic rats, hypothalamic POMC mRNA expression (measured by in situ hybridization) was reduced by 80% (P < 0.005), whereas AgRP mRNA levels were increased by 60% (P < 0.01), suggesting a marked decrease of hypothalamic melanocortin signaling. The change in POMC, but not in A g R P, mRNA levels was partially reversed by insulin treatment. By comparison, the effects of diabetes to increase hypothalamic neuropeptide Y (NPY) expression and to decrease corticotropin-releasing hormone (CRH) expression were normalized by insulin treatment, whereas the expression of mRNA encoding the long form of the leptin receptor in the arcuate nucleus was unaltered by diabetes or insulin treatment. UCP-3 mRNA expression in gastrocnemius muscle from diabetic rats was increased fourfold (P < 0.005), and the increase was prevented by insulin treatment. The effect of uncontrolled diabetes to decrease POMC, while increasing AgRP gene expression, suggests that reduced hypothalamic melanocortin signaling, along with increased NPY and decreased CRH signaling, could contribute to diabetic hyperphagia. These responses, in concert with increased muscle UCP-3 expression, may also contribute to the catabolic effects of uncontrolled diabetes on fuel metabolism in peripheral tissues. D i a b e t e s 49:244-252, 2000 H yperphagia (1,2) and altered fuel metabolism (3,4) are prominent features of uncontrolled diabetes. Although the pathogenesis of diabetic hyperphagia is incompletely understood, several observations suggest a key role for reduced signaling by insulin and leptin, hormones involved in the regulation of energy balance via their effects on the central nervous system (CNS) to regulate ...

A major paradigm in the field of obesity research is the existence of an adipose tissue-brain endocrine axis for the regulation of body weight. Leptin, the peptide mediator of this axis, is secreted by adipose cells. It lowers food intake and body weight by acting in the hypothalamus, a region expressing an abundance of leptin receptors and a variety of neuropeptides that influence food intake and energy balance. Among the most promising candidates for leptin-sensitive cells in the hypothalamus are arcuate nucleus neurons that co-express the anabolic neuropeptides, neuropeptide Y (NPY) and agouti-related peptide (AGRP), and those that express proopiomelanocortin (POMC), the precursor of the catabolic peptide, alphaMSH. These cell types contain mRNA encoding leptin receptors and show changes in neuropeptide gene expression in response to changes in food intake and circulating leptin levels. Decreased leptin signaling in the arcuate nucleus is hypothesized to increase the expression of NPY and AGRP. Levels of leptin receptor mRNA and leptin binding are increased in the arcuate nucleus during fasting, principally in NPY/AGRP neurons. These findings suggest that changes in leptin receptor expression in the arcuate nucleus are inversely associated with changes in leptin signaling, and that the arcuate nucleus is an important target of leptin action in the brain.