Elsevier

Brain Research

Volume 791, Issues 1–2, 27 April 1998, Pages 157-166
Brain Research

Research report
Fos expression in the brain induced by peripheral injection of CCK or leptin plus CCK in fasted lean mice

https://doi.org/10.1016/S0006-8993(98)00091-2Get rights and content

Abstract

We previously reported a synergistic interaction between leptin and cholecystokinin (CCK) to reduce food intake through CCK-A receptors in lean mice fasted for 24 h. To identify the activated neuronal pathways, we investigated changes in Fos expression in brain nuclei 2 h after single or combined intraperitoneal (i.p.) injections of leptin (120 μg/kg) and sulfated CCK-8 (3.5 μg/kg) in male lean mice (C57BL/6) fasted for 24 h using immunohistochemistry for Fos, the protein product of the early gene, c-fos. Leptin did not increase Fos expression in the brain compared with vehicle-treated mice. CCK increased the numbers of Fos-positive neurons in the nucleus of the solitary tract (NTS)/area postrema (AP), central nucleus of the amygdala (CeA) and, to a smaller extent, in the paraventricular nucleus of the hypothalamus (PVN) (5.2-, 2.3- and 0.3-fold respectively). Injections of leptin–CCK further enhanced Fos expression by 40% in the PVN compared with that induced by CCK alone, but not in the other nuclei. Devazepide (a CCK-A receptor antagonist, 1 mg/kg, i.p.) prevented the increase in Fos expression induced by leptin–CCK in the PVN and by CCK alone in the PVN, CeA and NTS/AP. These results indicate that in fasted mice, i.p. injection of CCK increases Fos expression in specific brain nuclei through CCK-A receptors while leptin alone had no effect. Leptin in conjunction with CCK selectively enhanced Fos expression in the PVN. The PVN may be an important site mediating the synergistic effect of leptin–CCK to regulate food intake.

Introduction

Leptin, a protein synthesized exclusively by the adipose tissue, is secreted into the circulation and plays a critical role in the long-term regulation of ingestive behavior and energy expenditure [10]. Convergent studies indicate that circulating leptin exerts its action directly in the central nervous system. Leptin-induced reduction of food intake had a delayed onset (4–5 h) and lower potency when injected peripherally compared with intracerebroventricularly (30 min onset) in lean or ob/ob mice or rats 4, 5, 10, 15, 37, 47, 48, 52. Circulating leptin is transported, through a saturable system, intact from the blood into selective brain areas 3, 22, 56and specific receptors are found in the choroid plexus and the hypothalamus 24, 33, 34, 51.

Cholecystokinin (CCK) is one of the gut peptides released by ingested food acting as a short-term signal to limit meal size [13]. One mechanism subserving the satiety effect of peripheral injection of CCK involves activation of CCK-A receptors located on vagal afferent pathways 38, 39, 62relaying information to specific brain sites regulating food intake 7, 12, 16, 21, 28. We recently reported a synergistic interaction between leptin and CCK to induce an early satiety signal as shown by the 80% suppression of food intake during the first 3 h after acute intraperitoneal injections of CCK and leptin at doses inefficient when given singly in 24-h fasted lean mice [4]. Similar leptin–CCK potentiation to inhibit daily caloric intake has been reported in another mice strain [30]. The leptin–CCK effect is mediated by CCK-A receptors and associated with enhanced Fos expression in the PVN 2 h post-injection in 24-h fasted mice [4].

The influence of an acute peripheral injection of leptin on Fos expression in the brain has not been studied in lean mice except in the PVN 4, 60and that of CCK has been extensively studied only in rats 16, 28, 43. The area postrema, nucleus tractus solitarius (NTS), supraoptic nucleus, PVN and central nucleus of the amygdala (CeA) expressed Fos in response to peripheral injection of CCK in rats 16, 28, 43and these sites may participate in the satiety effect of peripherally administered CCK 14, 17, 54.

To gain insight into neuronal mechanisms underlying the synergistic interaction between leptin and CCK, we examined first, the neuronal circuitry activated by single or combined intraperitoneal administrations of leptin and CCK in fasted lean mice. We focused on hindbrain nuclei (NTS and area postrema, AP) [41], limbic (CeA) and hypothalamic (arcuate nucleus) nuclei providing afferent inputs to the PVN 23, 29, 42, 44, 50and known to be involved in the regulation of food intake 14, 17, 31, 35, 54. Secondly, we investigated whether CCK action alone or combined with leptin is mediated by CCK-A receptors. Leptin and CCK were used at an intraperitoneal dose previously reported to be subthreshold when given singly to influence significantly hourly food intake in lean fasted mice [4].

Section snippets

Animals

Male lean mice (C57BL/6, +/+, 6–7-week-old, 20–25 g) (Harlan Laboratories, San Diego, CA) were housed under conditions of controlled temperature (21±1°C), humidity (30–35%) and lighting (0600–1800 h), with standard Purina Laboratory Chow and tap water ad libitum. All experiments were performed between 0830 h and 1300 h and approved by the Veterans Administration Animal Care and Use Committee (Animal Component of Research Protocol number 97-02-01).

Reagents and treatments

Aliquots of recombinant murine leptin (Amgen,

Results

Fasted mice injected i.p. twice with vehicles (saline) showed a low level of Fos expression (25±2 cells/section) in the NTS (Fig. 1Fig. 2A) similar to fasted mice with no i.p. injections (17±1 cells/section) (Fig. 1). CCK (3.5 μg/kg) or leptin (120 μg/kg) plus CCK increased the number of Fos-positive neurons in the NTS by 5.2 (156±2 cells/section) and 7.2 (204±8 cells/section) fold respectively, compared with vehicles (Fig. 1, Fig. 2C, D). Leptin alone had no effect (30±2 cells/section) as

Discussion

The present data show the effects of peripheral injection of leptin and CCK alone or in combination on Fos expression in the brains of fasted lean mice. In agreement with our previous report using a similar experimental design [4], we observed that leptin (120 μg/kg) injected i.p. with CCK (3.5 μg/kg) increased Fos expression in the PVN by 71% as monitored 2-h after injection in 24-h fasted lean mice pretreated with vehicle. This increase reflects a specific synergistic interaction between CCK

Conclusion

In summary, the present study shows that 24-h fasting in lean mice induces intense Fos expression in the arcuate nucleus, DMH and, to a lesser extent, the PVN which is not altered by an i.p. injection of leptin at a dose biologically active to reduce ingestive behavior after a 5-h food exposure 4, 5. CCK injected i.p. at a dose shown to be subthreshold to significantly reduce the 1 h cumulative food intake, elicits Fos expression mainly in the NTS/AP and in the CeA and, to a lesser extent, in

Acknowledgements

We thank Amgen Biologicals for providing the mouse recombinant leptin, and Mr. Paul Kirsch for helping in the preparation of the manuscript. This work was supported by the Research Scientist Award Grant MH 00663, the National Institute of Diabetes and Digestive and Kidney Diseases, Grants DK 30110 and DK 41301 (Animal Model Core). Dr. M.D. Barrachina was supported in part by a fellowship from Esplugues Foundation (Valencia, Spain).

References (62)

  • J.G. Mercer et al.

    Localization of leptin receptor mRNA and the long form splice variant (Ob–Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization

    FEBS Lett.

    (1996)
  • J.G. Mercer et al.

    Regulation of leptin receptor and NPY gene expression in hypothalamus of leptin-treated obese (ob/ob) and cold-exposed lean mice

    FEBS Lett.

    (1997)
  • F.J. Minano et al.

    GABAA receptors in the amygdala: role in feeding in fasted and satiated rats

    Brain Res.

    (1992)
  • A.M. Mistry et al.

    Elevated neuronal c-fos-like immunoreactivity and messenger ribonucleic acid (mRNA) in genetically obese (ob/ob) mice

    Brain Res.

    (1994)
  • A.M. Mistry et al.

    Leptin rapidly lowers food intake and elevates metabolic rates in lean and ob/ob mice

    J. Nutr.

    (1997)
  • K. Miyasaka et al.

    Lack of satiety effect of cholecystokinin (CCK) in a new rat model not expressing the CCK-A receptor gene

    Neurosci. Lett.

    (1994)
  • B.R. Olson et al.

    Cholecystokinin induces c-fos expression in hypothalamic oxytocinergic neurons projecting to the dorsal vagal complex

    Brain Res.

    (1992)
  • T. Petrov et al.

    Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat

    Brain Res.

    (1993)
  • L.A. Tartaglia et al.

    Identification and expression cloning of a leptin receptor, OB-R

    Cell

    (1995)
  • D. Van der Kooy

    Area postrema: site where cholecystokinin acts to decrease food intake

    Brain Res.

    (1984)
  • B. Xu et al.

    Neuropeptide Y injection into the fourth cerebroventricle stimulates c-fos expression in the paraventricular nucleus and other nuclei in the forebrain: effect of food consumption

    Brain Res.

    (1995)
  • R.S. Ahima et al.

    Role of leptin in the neuroendocrine response to fasting

    Nature

    (1996)
  • M.D. Barrachina et al.

    Synergistic interaction between leptin and cholecystokinin to reduced short-term food intake in lean mice

    Proc. Natl. Acad. Sci. U.S.A.

    (1997)
  • M.D. Barrachina et al.

    Leptin-induced decrease in food intake is not associated with changes in gastric emptying in lean mice

    Am. J. Physiol.

    (1997)
  • L.S. Brady et al.

    Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats

    Neuroendocrinology

    (1990)
  • K.M. Buller et al.

    Involvement of medullary catecholamine cells in neuroendocrine responses to systemic cholecystokinin

    J. Neuroendocrinol.

    (1996)
  • L.A. Campfield et al.

    Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks

    Science

    (1995)
  • S. Ceccatelli et al.

    Expression of c-fos immunoreactivity in transmitter-characterized neurons after stress

    Proc. Natl. Acad. Sci. U.S.A.

    (1989)
  • I. Cusin et al.

    The weight-reducing effect of an intracerebroventricular bolus injection of leptin in genetically obese fa/fa rats. Reduced sensitivity compared with lean animals

    Diabetes

    (1996)
  • H.E.W. Day et al.

    Evidence that cholecystokinin induces immediate early gene expression in the brainstem, hypothalamus and amygdala of the rat by a CCKA receptor mechanism

    Neuropharmacology

    (1994)
  • G.L. Edwards et al.

    Dorsomedial hindbrain participation in cholecystokinin-induced satiety

    Am. J. Physiol.

    (1986)
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