Negative Energy Balance Blocks Neural and Behavioral Responses to Acute Stress by "Silencing" Central Glucagon-Like Peptide 1 Signaling in Rats

Abstract

Previous reports indicate that caloric restriction attenuates anxiety and other behavioral responses to acute stress, and blunts the ability of stress to increase anterior pituitary release of adrenocorticotropic hormone. Since hindbrain glucagon-like peptide-1 (GLP-1) neurons and noradrenergic prolactin-releasing peptide (PrRP) neurons participate in behavioral and endocrine stress responses, and are sensitive to the metabolic state, we examined whether overnight food deprivation blunts stress-induced recruitment of these neurons and their downstream hypothalamic and limbic forebrain targets. A single overnight fast reduced anxiety-like behavior assessed in the elevated-plus maze and acoustic startle test, including marked attenuation of light-enhanced startle. Acute stress [i.e., 30 min restraint (RES) or 5 min elevated platform exposure] robustly activated c-Fos in GLP-1 and PrRP neurons in fed rats, but not in fasted rats. Fasting also significantly blunted the ability of acute stress to activate c-Fos expression within the anterior ventrolateral bed nucleus of the stria terminalis (vlBST). Acute RES stress suppressed dark-onset food intake in rats that were fed ad libitum, whereas central infusion of a GLP-1 receptor antagonist blocked RES-induced hypophagia, and reduced the ability of RES to activate PrRP and anterior vlBST neurons in ad libitum-fed rats. Thus, an overnight fast "silences" GLP-1 and PrRP neurons, and reduces both anxiety-like and hypophagic responses to acute stress. The partial mimicking of these fasting-induced effects in ad libitum-fed rats after GLP-1 receptor antagonism suggests a potential mechanism by which short-term negative energy balance attenuates neuroendocrine and behavioral responses to acute stress.

Significance statement: The results from this study reveal a potential central mechanism for the "metabolic tuning" of stress responsiveness. A single overnight fast, which markedly reduces anxiety-like behavior in rats, reduces or blocks the ability of acute stress to activate hindbrain neurons that are immunoreactive for either prolactin-releasing peptide or glucagon-like peptide 1, and attenuates the activation of their stress-sensitive projection targets in the limbic forebrain. In nonfasted rats, central antagonism of glucagon-like peptide 1 receptors partially mimics the effect of an overnight fast by blocking the ability of acute stress to inhibit food intake, and by attenuating stress-induced activation of hindbrain and limbic forebrain neurons. We propose that caloric restriction attenuates behavioral and physiological responses to acute stress by "silencing" central glucagon-like peptide 1 signaling pathways.

Keywords: anxiety; fasting; food deprivation; hypophagia; nucleus of the solitary tract.

Figure 1.

Overnight fasting attenuates anxiety-like behavior in the EPMZ. A, Group summary data depicting time spent in the open versus the closed arms of the EPMZ by ad libitum-fed (n = 6) or fasted rats (n = 6). B, Group summary data depicting the number of open arm versus closed arm entries by ad libitum-fed or fasted rats. Asterisks indicate significant differences between ad libitum-fed and fasted groups. *p < 0.05. ad lib, Ad libitum .

Figure 2.

Overnight fasting reduces acoustic startle amplitude and attenuates light-enhanced startle. A, Group summary data depicting peak startle amplitudes at each noise intensity (90, 95, or 105 dB) in ad libitum-fed (n = 8) versus overnight-fasted rats (n = 8) tested in Phase I “dark” versus Phase II “light” conditions. *Startle amplitude is significantly elevated in light versus dark testing conditions at all three noise intensity levels in fed rats, but only at 95 and 105 dB in fasted rats (within-feeding group comparisons, p < 0.05); @under both lighting conditions, startle amplitudes at each noise intensity level are significantly lower in fasted versus fed rats, p < 0.05. B, Group summary data depicting the magnitude of light-enhanced startle, calculated as the percentage increase in startle amplitude in Phase II (light) versus Phase I (dark) conditions at each noise intensity level. @The magnitude of light-enhanced startle is significantly lower in fasted versus fed rats at each noise intensity level, p < 0.05.

Figure 3.

Fasting eliminates c-Fos activation within cNTS and medullary reticular GLP-1 neurons at baseline (nonhandled) and after acute stress. A–D, Representative color images depicting neuronal c-Fos expression (black nuclear label) within GLP-1⁺ neurons (brown cytoplasmic label) in the cNTS (A, C) and reticular formation (B, D) in rats exposed to elevated platform stress. A, Robust c-Fos activation within the cNTS (∼14.36 mm caudal to bregma) in an ad libitum-fed rat. Inset, Higher-magnification view of several c-Fos⁺ GLP-1 neurons. B, c-Fos⁺/GLP-1⁺ neurons within the reticular formation in an ad libitum-fed rat. C, cNTS GLP-1⁺ neurons do not express c-Fos in a fasted rat after elevated platform exposure. Inset, Higher-magnification view of several GLP-1⁺ neurons, none of which express c-Fos. D, Reticular GLP-1 neurons do not express c-Fos in a fasted rat after elevated platform exposure. E, Summary data illustrating the proportion of double-labeled neurons (i.e., both GLP-1 and c-Fos⁺) within the cNTS and adjacent reticular formation in ad libitum-fed rats (solid bars) or fasted rats (DEP; open bars) after no manipulation (nonhandled), after restraint stress, or after elevated platform stress. See Table 1 for two-way ANOVA results. Fasting completely eliminated c-Fos expression by GLP-1 neurons. Asterisks indicate significantly reduced (*p < 0.05) GLP-1 c-Fos activation in DEP rats versus ad libitum-fed rats within the same treatment group. Within the same feeding status group (i.e., ad libitum or DEP), bars with different letters (i.e., A, B, C) are significantly different (p < 0.05), whereas bars with the same letter (a) are not. ad lib, Ad libitum; cc, central canal. Scale bars: (in A) A, C, 200 μm; (in B) B, D, 50 μm.

Figure 4.

Overnight fasting (DEP) reduces the activation of noradrenergic PrRP⁺ and PrRP⁻ A2 neurons at baseline and after acute stress. A, B, Representative color images of sections through the cNTS (∼14.36 mm caudal to bregma) from two rats exposed to restraint stress. Sections are triple labeled for neuronal c-Fos expression (blue nuclear label), PrRP (red), and DβH (green). All PrRP⁺ neurons are also DβH⁺ and thus appear yellow/orange. Green cells are DβH⁺/PrRP⁻ A2 neurons. A, Restraint robustly activates cNTS c-Fos expression in an ad libitum-fed rat, including activation of most PrRP⁺/DβH⁺ neurons and a smaller proportion of PrRP⁻/DβH⁺ neurons. Inset, Higher-magnification view of several c-Fos⁺/PrRP⁺/DβH⁺ neurons. B, Restraint activates relatively few PrRP⁺/DβH⁺ or PrRP⁻/DβH⁺ neurons in a fasted rat. Inset, Higher-magnification view of several PrRP⁺/DβH⁺ and PrRP⁻/DβH⁺ neurons, some of which express c-Fos. C, D, Summary data reporting the proportion of PrRP⁺/DβH⁺ neurons expressing c-Fos (C), or PrRP⁻/DβH⁺ neurons expressing c-Fos (D) in ad libitum-fed rats (solid bars) or fasted rats (DEP; open bars) after no manipulation (nonhandled; n = 6 ad libitum, n = 7 DEP), restraint stress (n = 4 ad libitum, n = 8 DEP), or elevated platform stress (n = 6 ad libitum, n = 6 DEP). C, DEP eliminated c-Fos expression by PrRP⁺/DβH⁺ neurons in nonhandled control rats or in rats after elevated platform stress and significantly reduced c-Fos expression by PrRP⁺/DβH⁺ neurons in rats exposed to restraint. D, Relatively few PrRP⁻/DβH⁺ neurons expressed c-Fos in nonhandled control rats, regardless of feeding status. Fasting attenuated stress-induced c-Fos expression in PrRP⁻/DβH⁺ neurons. See Table 1 for two-way ANOVA results. In C and D, asterisks indicate significantly reduced (*p < 0.05) neural c-Fos expression in DEP versus ad libitum-fed rats in the same stress group. Within the same feeding status group (i.e., ad libitum or DEP), bars with different letters (i.e., A, B, C or a, b, c) are significantly different (p < 0.05). ad lib, Ad libitum; cc, central canal. Scale bar: (in A) A, B, 200 μm.

Figure 5.

Summary data illustrating the number of c-Fos⁺ neurons within the GLP-1 terminal-rich region of the mpPVN in ad libitum-fed rats (solid bars) or fasted rats (DEP; open bars) after no manipulation (nonhandled; n = 6 ad libitum, n = 7 DEP), restraint stress (n = 4 ad libitum, n = 8 DEP), or elevated platform stress (n = 6 ad libitum, n = 6 DEP). Compared with nonhandled control rats, either restraint or elevated platform exposure increased c-Fos expression within the mpPVN, regardless of feeding status. Despite the apparent trend toward reduced mpPVN c-Fos activation in fasted rats under nonhandled control conditions or after elevated platform exposure, there was no significant main effect of feeding status, and no interaction between feeding status and stress treatment on mpPVN c-Fos activation (see Table 1 for two-way ANOVA results). ad lib, Ad libitum.

Figure 6.

Fasting reduces c-Fos activation within the anterior vlBST after acute stress. A, B, Representative color images depicting neuronal c-Fos expression (blue nuclear label) within the coextensive PrRP (red) and DβH (green) terminal-rich region of the vlBST (∼0.26 mm caudal to bregma). A, In an ad libitum-fed rat, many neurons within and surrounding the vlBST express c-Fos after restraint. B, Fewer vlBST neurons express c-Fos in a fasted rat after restraint. C, Summary data reporting the number of c-Fos⁺ neurons per 100 μm² area of the PrRP/DβH terminal-rich vlBST in ad libitum-fed rats (solid bars) or fasted rats (DEP; open bars) after no manipulation (nonhandled; n = 6 ad libitum, n = 7 DEP), restraint stress (n = 4 ad libitum, n = 8 DEP), or elevated platform stress (n = 6 ad libitum, n = 6 DEP). See Table 1 for two-way ANOVA results. Fasting did not affect baseline c-Fos, but significantly attenuated stress-induced c-Fos expression in the vlBST. Asterisks indicate significantly reduced (*p < 0.05) vlBST neural c-Fos expression in DEP rats versus ad libitum-fed rats in the same treatment group. Within the same feeding status group (i.e., ad libitum or DEP), bars with different letters (i.e., A, B, C or a, b, c) are significantly different (p < 0.05). AC, Anterior commissure; ad lib, Ad libitum. Scale bar: (in A) A, B, 200 μm.

Figure 7.

The ability of restraint stress to suppress dark-onset food intake in ad libitum-fed rats is blocked by lateral intracerebroventricular administration of a specific GLP-1 receptor antagonist (Ex9). Chow intake (expressed as a percentage of body weight) is illustrated from 0 to 30 min, from 30 to 60 min, and cumulatively (0–60 min) at baseline (n = 24), and after each experimental treatment (n = 6/group). Control intracerebroventricular infusion of saline vehicle or Ex9 alone did not alter chow intake. After intracerebroventricular infusion of saline, restraint stress significantly reduced food intake during the first 30 min. This hypophagic effect was not compensated for during the second 30 min period, such that the cumulative 60 min intake remained suppressed. Pretreatment with intracerebroventricular injection of Ex9 significantly attenuated restraint stress-induced hypophagia during the first 30 min and at the cumulative 60 min time point. *Significantly less intake (p < 0.05) compared with baseline and compared with intake by rats in all other treatment groups within the same time bin.

Figure 8.

Lateral intracerebroventricular infusion of a GLP-1 receptor antagonist (Ex9) significantly reduces the ability of RES stress to activate PrRP⁺ cNTS neurons and neurons within the anterior vlBST in ad libitum-fed rats. A, The majority of PrRP⁺ neurons (brown) express c-Fos (black nuclei) after RES in rats pretreated with intracerebroventricular injection of saline (SAL). B, RES-induced c-Fos within the PrRP-rich anterior vlBST in a rat treated with intracerebroventricularly injected saline after RES. C, RES-induced activation of PrRP neurons is reduced in rats pretreated with intracerebroventricular injection of Ex9. D, RES-induced activation of neurons within the PrRP-rich anterior vlBST is reduced in rats pretreated with intracerebroventricular injection of Ex9. E, Quantification of RES-induced activation of PrRP and GLP-1 neurons in rats pretreated with intracerebroventricular injection of either saline (n = 5) or Ex9 (n = 8); *p < 0.05, intracerebroventricular saline vs Ex9. F, Quantification of RES-induced activation within the anterior vlBST in rats pretreated intracerebroventricularly with either saline (n = 5) or Ex9 (n = 8); *p < 0.05, intracerebroventricularly injected saline vs Ex9. Scale bars: (in C, D) A–D, 200 μm.