Figure 1.
Schematic of proSAAS and proSAAS-derived peptides, and the localization of proSAAS, proNPY, POMC, and PC1/3 mRNA in the hypothalamus of the mouse.
Top panel: Schematic diagram showing the major proSAAS-derived peptides previously detected in various brain regions and other relevant peptides. The relative size and position of these peptides within proSAAS are indicated. Bottom panels: Images of proSAAS, proNPY, POMC, and PC1/3 mRNA distribution were downloaded from the Allen Mouse Brain Atlas [http://mouse.brain-map.org], Seattle, WA, Allen Institute for Brain Science ©2009 [50].
Figure 2.
Coronal sections through mouse hypothalamus showing immunofluorescence colocalization of peptides in the arcuate nucleus.
The upper panels show immunoreactive PEN/LEN peptides in red and immunoreactive big LEN peptides in green. The merged images (right) show general colocalization of the two peptides. The panels in the second row show immunoreactive PEN/LEN peptides in red and immunoreactive NPY peptides in green, and merged images show essentially complete colocalization of the two peptides. The panels in the third row show immunoreactive PEN/LEN peptides in red and immunoreactive α-MSH peptides in green. Merged images show no colocalization of the two peptides. The bottom row shows immunoreactive big LEN peptides in red and immunoreactive α-MSH peptides in green. Merged images show no colocalization of the two peptides. Nuclei are stained blue using DAPI (4,6- diamidino-2-phenylindole). The scale bar indicates 10 µm.
Figure 3.
Coronal sections through the hypothalamus of NPY-GFP mice showing immunofluorescence localization of peptides in the arcuate nucleus.
The upper panel shows immunoreactive big LEN peptides in red and NPY neurons expressing GFP in the cytosol in green. In the merged images big LEN peptide immunoreactivity can be seen in the green GFP-expressing neurons. The middle row of panels shows immunoreactive PEN peptides in red and NPY neurons in green. In the merged images, PEN peptide immunoreactivity can be seen in the green GFP-expressing neurons. The bottom panels show a section incubated with pre-immune serum and secondary Cy3 antibodies, with background staining only. Nuclei are stained blue using DAPI (4,6- diamidino-2-phenylindole). Scale bar 10 µm.
Figure 4.
Levels of proSAAS mRNA and proNPY mRNA in subhypothalamic brain regions.
Left: ProSAAS mRNA in the MBH, PVN, and LH in wild-type mice. Right: The MBH expresses the highest levels of proNPY mRNA, while the PVN and LH express much lower levels. Error bars show standard error of the mean (n = 8).
Figure 5.
Comparison of relative levels of proSAAS mRNA and proNPY mRNA in wild-type versus Cpefat/fat mice.
A: proSAAS mRNA levels are significantly lower (p = 0.041) in the MBH of the Cpefat/fat mice, relative to wild-type (WT) mice, and show a tendency to decrease in the LH of the Cpefat/fat mice, but in this brain region the difference is not statistically significant (p = 0.23). B: proNPY mRNA levels show a tendency to decrease in the MBH and LH of Cpefat/fat mice, relative to wild-type mice, but these changes are not statistically significant (p = 0.17 and 0.19, respectively). Error bars show standard error of the mean for n = 8. Abbreviations: WT, wild-type; fat/fat, Cpefat/fat. *, p<0.05 using Student's two-tailed t-test.
Figure 6.
Comparison of proSAAS mRNA and proNPY mRNA levels in fed and fasted mice in various subhypothalamic brain regions.
A. ProSAAS mRNA levels do not show a significant change in fasted WT mice relative to mice fed ad libitum in any of the brain regions examined. B. ProNPY mRNA levels increase significantly in the MBH of fasted mice relative to fed mice, but are not affected in the other two brain regions examined. C. ProSAAS mRNA levels do not show a significant change with fasting in subhypothalamic brain regions of Cpefat/fat mice. D. ProNPY mRNA is significantly increased in the MBH of fasted Cpefat/fat mice. Error bars show standard error of the mean for n = 8 in WT mice and n = 3 in Cpefat/fat mice. *, p<0.05 using Student's two-tailed t-test.
Table 1.
Relative levels of proSAAS-derived peptides in fed and fasted mice.
Figure 7.
Comparison of food intake in mice injected with proSAAS-derived peptides.
Mice do not show a significant peptide-induced change in food intake measured 1 hour, 2 hours, or 14 hours after the injection of either saline or the proSAAS-derived peptides: PEN, big LEN, little SAAS, or a combination of PEN and big LEN. Altogether 18 mice were cannulated and after recovery from surgery, injected with either 10 µg of peptide in saline or with saline alone. After 3–7 days of recovery, the mice were re-tested with the saline and peptide groups switched so that over the course of the study, each mouse received at least one control saline injection and injection of at least one of the different peptides. Error bars show standard error of the mean for saline (n = 37); PEN (n = 15); big LEN (n = 15); little SAAS (n = 8); and the combination of PEN and big LEN (n = 16).
Figure 8.
Comparison of food intake in mice injected with purified antibodies directed against proSAAS-derived peptides.
Altogether, 18 mice were cannulated and in each experiment the mice were injected with either saline, control antibody (Cont Ab), or one of the antibodies directed against proSAAS-derived peptides, and food intake was measured 1 hour, 2 hours, and 14 hours after the injection. The antibody to PEN significantly reduced food intake at all time points examined. The antibody to big LEN showed a significant decrease in food intake after 1 and 2 hours, but not after 14 hours. In contrast, the antibody to little LEN did not significantly affect food intake at any time point examined, and the antibody to little SAAS showed a slight increase after 1 hour, but not at the other time points. Error bars show the standard error of the mean; saline (n = 21); control Ab (n = 19); PEN Ab (n = 8); big LEN Ab (n = 20); little LEN Ab (n = 10); little SAAS Ab (n = 8). *, p<0.05, **, p<0.01, ***, p<0.001 relative to control Ab, using Student's two-tailed t-test.
Figure 9.
Big LEN suppresses excitatory synaptic input to PVN parvocellular neurons via the release of a retrograde messenger.
A. Whole-cell patch clamp recording of spontaneous synaptic currents in a parvocellular neuroendocrine cell in the PVN in the absence (Control) and presence of big LEN (Big LEN). Big LEN (1 µM) caused a decrease in the frequency of negative synaptic currents, which are EPSCs mediated by glutamate release. B. Cumulative frequency plots of EPSC interevent intervals and EPSC amplitudes taken from the recording shown in A. Big LEN caused a shift toward longer interevent intervals, indicative of a decrease in EPSC frequency, but had no effect on the cumulative amplitude distribution. C. Mean frequency, amplitude and decay time of EPSCs in big LEN as a percentage of control measures. Big LEN (1 µM) decreased the frequency of EPSCs, but had no effect on EPSC amplitude or decay time (n = 9), suggesting a presynaptic suppression of glutamate release, but no effect on postsynaptic glutamate sensitivity. D. The effect of big LEN was maintained in TTX, indicating that the effect was action potential independent, but was blocked by an inhibitor of G protein activity (GDP-β-S) applied via the patch pipette into the postsynaptic cell, suggesting that the effect is mediated by the activation of a postsynaptic G protein-coupled receptor and release of a retrograde messenger.