Divergent leptin signaling in proglucagon neurons of the nucleus of the solitary tract in mice and rats

Abstract

The central targets mediating the anorectic and other actions of leptin have yet to be fully identified. Although previous studies focused on the hypothalamus, leptin also acts on neurons in extrahypothalamic sites, including the nucleus of the solitary tract (NTS). Moreover, injection of leptin into the NTS of rats suppresses food intake. Within the central nervous system, glucagon-like peptide (GLP-1), a product of proglucagon, is synthesized almost exclusively in neurons of the NTS. Intracerebroventricular administration of GLP-1 inhibits energy intake, and GLP-1 receptor antagonists attenuate the anorexic effects of leptin in rats. To examine whether NTS proglucagon neurons are directly regulated by leptin, we performed double GLP-1 and phosphorylation of signal transducer and activator of transcription-3 immunohistochemistry on brain sections from ip leptin-treated mice and rats. Leptin induced phosphorylation of signal transducer and activator of transcription-3 in 100% of GLP-1 cells in the caudal brainstem of mice. In striking contrast, 0% of GLP-1-positive neurons in rats responded to leptin. We then measured regulation of NTS proglucagon mRNA using real-time RT-PCR in mice and rats fed ad libitum, fasted, or fasted and treated ip with leptin. In mice, proglucagon mRNA fell by fasting, and this was prevented by leptin administration. In rats, by contrast, proglucagon mRNA was unaffected by either fasting or leptin. Taken together, our studies reveal direct regulation of proglucagon neurons by leptin in mice but not rats along with corresponding species differences in the regulation of proglucagon mRNA expression. These data, combined with previous results, suggest a different mechanism of interaction between leptin and NTS proglucagon neurons in mice and rats.

Figure 1

Anatomical localization of leptin-responsive neurons in the NTS of rats and mice. Male Sprague Dawley rats and C57BL/6 mice were injected with leptin ip or PBS for 45 min. Series of coronal hindbrain sections were subjected to P-STAT3 DAB IHC. Shown are representative photomicrographs of P-STAT3 DAB IHC in the NTS of leptin-treated rats (top row) and leptin-treated mice (third row). A matched series of PBS-treated rats and mice were, respectively, shown in the second row and bottom row. All sections are ordered in a rostral to caudal manner (see Bregma levels). cc, Central canal; Cu, cuneate nucleus; Gr, gracile nucleus. Scale bar, 200 μm.

Figure 2

Anatomical localization of GLP-1-producing neurons in the NTS of rats and mice. Shown are representative images of GLP-1 fluorescence IHC in hindbrain sections from rats (A) and mice (B). All sections are ordered in a rostral to caudal manner (see Bregma levels). The last row of images represent higher magnifications of the boxed areas above. cc, Central canal; Cu, cuneate nucleus; Gr, gracile nucleus. Scale bar, 200 μm.

Figure 3

Leptin induces P-STAT3 in 100% GLP-1-positive cells in the NTS of mice but not rats. Male Sprague Dawley rats or C57BL/6 mice were injected with leptin ip for 45 min. Series of coronal brain sections were subjected to combined P-STAT3 DAB and GLP-1 fluorescence double IHC. A and B, Shown are three representative merged photomicrographs from double IHC analyses of rats and mice, respectively. All sections are ordered in a rostral to caudal manner (from top to bottom). C, Shown are high magnifications (top, P-STAT3 DAB IHC; middle, GLP-1 green fluorescence IHC; bottom, merged photomicrograph from the double IHC) of the area marked in bottom of B. cc, Central canal; Cu, cuneate nucleus; Gr, gracile nucleus. Scale bars (A and B), 200 μm; (C), 50 μm.

Figure 4

Proglucagon mRNA is regulated by fasting and leptin in the NTS of mice but not rats. Male Sprague Dawley rats and C57BL/6 mice were fed ad libitum and treated with vehicle (PBS), fasted (48 h) and treated with vehicle (PBS), or fasted (48 h) and treated with leptin (2 mg/kg, two times daily, ip). Tissues from the caudal brainstem that included the entire NTS were dissected. Shown are real-time PCR results for proglucagon mRNA. Data are means ± sem, n = 10 in each mice group, n = 14 in each group of rats. *, P < 0.05; **, P < 0.01. NS, Not significant.