Activation of murine pre-proglucagon-producing neurons reduces food intake and body weight

Abstract

Peptides derived from pre-proglucagon (GCG peptides) act in both the periphery and the CNS to change food intake, glucose homeostasis, and metabolic rate while playing a role in anxiety behaviors and physiological responses to stress. Although the actions of GCG peptides produced in the gut and pancreas are well described, the role of glutamatergic GGC peptide-secreting hindbrain neurons in regulating metabolic homeostasis has not been investigated. Here, we have shown that chemogenetic stimulation of GCG-producing neurons reduces metabolic rate and food intake in fed and fasted states and suppresses glucose production without an effect on glucose uptake. Stimulation of GCG neurons had no effect on corticosterone secretion, body weight, or conditioned taste aversion. In the diet-induced obese state, the effects of GCG neuronal stimulation on gluconeogenesis were lost, while the food intake-lowering effects remained, resulting in reductions in body weight and adiposity. Our work suggests that GCG peptide-expressing neurons can alter feeding, metabolic rate, and glucose production independent of their effects on hypothalamic pituitary-adrenal (HPA) axis activation, aversive conditioning, or insulin secretion. We conclude that GCG neurons likely stimulate separate populations of downstream cells to produce a change in food intake and glucose homeostasis and that these effects depend on the metabolic state of the animal.

Figure 1. Characterization of Gcg-Cre mice.

(A) Cre recombinase was inserted into the Gcg gene locus of BAC RP23-242F22 at the ATG start codon. Red boxes, 5′ and 3′ untranslated regions; white boxes, Gcg exons; lines, intronic DNA. (B–I) Cre recombinase expression within GCG neurons was visualized by crossing Gcg-Cre mice with tdTomato reporter animals. tdTomato fluorescence (NTS in C; VLM in F) is restricted to GLP-1–expressing cells (B and E) in the NTS (D) and the VLM (G). Specificity of Cre recombinase expression is high, with virtually all tdTomato-labeled cells in the NTS (99%) and more than 92% in the VLM showing GLP-1 immunofluorescence (H). The majority of GLP-1–labeled neurons show Cre recombinase activity, with efficacy ranging from 84% in the VLM to 93% in the NTS (I). The few GLP-1–positive neurons that lack tdTomato signal are marked with green asterisks (in B, D, E, and G). (J and K) PVH and arcuate nuclei (Arc) neurons did not show expression, but tdTomato-labeled axons and terminals originating from brainstem GCG neurons were readily observed. AHE, anterior hypothalamic nucleus; ME, median eminence. (L–O) GCG-Gq DREADD transgenic mice exhibit normal body weight (L), daily chow food intake (M), and normal fed (N) and fasting (O) glucose levels. Scale bars in B–G, J, and K are in micrometers.

Figure 2. Activation of GCG neurons transfected with Gq DREADD.

Microinjection of AAV2/5-hSyn-DIO-hM₃Dq (Gq)-mCherry (A) resulted in Gq DREADD expression exclusively in GLP-1–immunoreactive neurons of the NTS (B) and the VLM (C); GLP-1, black precipitate; mCherry, brown precipitate. ITR, inverted terminal repeat. Some GLP-1–labeled cells lacked DsRed labeling (marked with asterisks in B). (D–F) Functional Gq DREADD expression was demonstrated by intense Fos induction within DsRed-labeled neurons of the NTS (D) and VLM (E) 2 hours following CNO injection. Strong Fos IR was seen in DsRed-labeled neurons (F). CNO treatment induced intense Fos staining largely limited to neurons displaying GLP-1 IR in the NTS (G) and the VLM (H). Most GCG neurons showed strong Fos staining in both NTS and VLM (I). Two saline-injected GCG-Gq DREADD mice showed a complete lack of Fos IR within DsRed- (J) and GLP-1–immunolabeled neurons (K). (L) Distribution of GCG neurons in the caudal medulla. (M–R) A small but significant increase in CNO-induced Fos staining was seen in the PVH (M, N, and O) and the arcuate nucleus (Arc; P, Q, and R), when GCG-Gq DREADD mice (M and P) were compared with controls (N and Q). n = 4 animals per group; all comparisons made using t test, *P < 0.05, **P < 0.01. Scale bars are in micrometers.

Figure 3. CNO application produces a small increase in GCG neuronal resting membrane potential and increases firing frequency following current injection.

CNO elevates resting membrane potential and potentiates action potential firing in mCherry-labeled neurons in the NTS following current injection. Recordings were made from tissue sections from 4 animals. (A) mCherry expression within the NTS. AP, area postrema; cc, central canal. (B) Patch clamped mCherry neuron. (C and D) Representative traces showing that bath application of CNO (9 μM; 10 minutes) evoked firing in mCherry-labeled neurons. (E) An expanded trace showing a single depolarizing event with spikes during perfusion of CNO (9 μM). Resting membrane potential was increased by 6 mV, an effect that led to a minimal increase in spontaneous firing rate. ACSF, artificial cerebrospinal fluid. (F) Labeled neurons did not fire action potentials in the absence of CNO. (G) CNO (9 μM) did not evoke spikes in unlabeled neurons. Breaks shown represent 2 minutes of duration. Spikes elicited by a depolarized current injection step to 110 pA in labeled neurons before (H) and after 10 minutes of CNO (9 μM) application (I). (J) Plot of firing frequency as a function of increasing depolarizing current steps shows a significant increase in firing frequency in labeled neurons after 10 minutes of CNO application (9 μM; 2-way repeated measures ANOVA, main effect of CNO treatment, F_1,10 = 7.782, P = 0.019). Spikes elicited by a depolarized current injection step to 110 pA in unlabeled neurons before (K) and (L) after 10 minutes of CNO (9 μM) application. (M) Plot of firing frequency as a function of increasing depolarizing current steps shows no change in firing frequency in unlabeled neurons after 10 minutes of CNO (9 μM; n = 5). Values represent mean ± SEM. *P < 0.05.

Figure 4. GCG neuron activation modulates food intake in both fed and fasted animals.

Activation of GCG neurons by CNO (2 mg/kg) reduced food intake both in the light phase (A, paired t test, n = 11, **P = 0.005) and during the first 3 hours of the dark phase (B, paired t test, n = 8, *P = 0.026) upon return of food 2 hours after i.p. CNO/saline injections. HCD intake during early daytime when mice were sated (following ad libitum overnight feeding on chow) was significantly reduced after CNO injection (C, 2-way repeated measures ANOVA, main effect of treatment F_1,11 = 8.66, *P = 0.0134). Food intake during daytime refeeding following 18 hours of fasting was also significantly reduced (D, 2-way repeated measures ANOVA, n = 13, main effect of treatment, F_1,12 = 5.118, *P = 0.0430). WT littermates that underwent sham brain surgery were tested by injection of either CNO (5 mg/kg) or saline i.p., and no effects of CNO were apparent during refeeding after fasting (E, 2-way repeated measures ANOVA, F_1,5 = 0.06308, P = 0.8) or during HCD feeding (F, 2-way repeated measures ANOVA, F_1,5 = 0.07558, P = 0.62).

Figure 5. GCG neuronal activation produces a selective effect on glucose homeostasis.

Insulin secretion and insulin sensitivity were not affected by CNO treatment (A, 2-way repeated measures ANOVA, F_1,11 = 0.04572, P = 0.8346; B, 2-way repeated measures ANOVA, F_1,15 = 0.4948, P = 0.4926). Fed-state blood glucose levels showed a modest increase 1 hour after CNO delivery as compared with saline treatment (C, paired t test, *P = 0.038). i.p. pyruvate tolerance test showed a reduction in gluconeogenesis (D, 2-way repeated measures ANOVA, significant effect of treatment, F_5,35 = 24.24, *P = 0.0001; E, area under the curve, paired t test, *P = 0.0304). 2-Deoxyglucose uptake assay showed no effect of CNO on glucose disposal (F, t test for each tissue sourced from saline- and CNO-treated animals, EDL t = 0.3492, P = 0.7388; TA t = 0.007964, P = 0.9939; soleus t = 0.893, P = 0.398; WAT t = 0.5955, P = 0.5732; liver t = 0.4717, P = 0.6538). Finally, fasted insulin levels were unchanged (G, t test, n = 8, t = 0.056, P = 0.956).

Figure 6. GCG neuronal activation alters metabolic rate and fasting locomotion, but has no effect on conditioned taste aversion, corticosterone levels, or anxiety-related behaviors.

(A–C) GCG stimulation produces a small change in energy metabolism, with no effect on carbohydrate and fat utilization. CNO injection increased metabolic rate during the 3 hours after dark onset (A, VO₂, paired t test n = 8, t = 2.661, *P = 0.0324; and VCO₂, paired t test t = 2.273, P = 0.05), with no change in RER (B, paired t test, t = 0.1026, P = 0.9212). Activity was reduced (beam break counts in C; paired t tests t = 3.12 and 3.18, *P = 0.017 and 0.016, for X and Y ambulations, respectively). (D) Ucp1, Pparg, and Pgc1a gene expression was unchanged in iBAT dissected from CNO- and saline-treated animals Ucp1 (t test t = 0.94, P = 0.398), Pparg (t test, t = 0.462, P = 0.66), Pgc1a (t test, t = 0.07, P = 0.94). (E) Fourteen days of HCD feeding followed by CNO injections every 8 hours for 48 hours did not produce a change in body weight (2-way repeated measures ANOVA, F_1,6 = 1.336, P = 0.292). (F) CNO did not affect saccharin preference (paired t test, t = 0.7649, P = 0.4733). Positive control LiCl produced a strong conditioned taste aversion (paired t test, t = 5.816, P = 0.0011). (G) CNO did not increase serum corticosterone levels at 70 and 90 minutes, despite producing a reduction in food intake (shown in Figure 4A) (ANOVA, F_2,35 = 0.1134, P = 0.3834). (H–J) CNO injection did not change performance in the elevated plus maze (H, t test, open arms t = 0.7684, P = 0.4534; closed arms t = 0.6968, P = 0.4954), open field (I, time spent in open field, t test, center t = 0.1924, P = 0.8509, border t = 0.3785, P = 0.7123; J, locomotor activity reflected in distance traveled in both border and central zones, t test, border t = 0.5473, P = 0.5991, central t = 0.02015, P = 0.9844).

Figure 7. Anorexigenic effect of GCG neuronal stimulation is enhanced in DIO Gcg-Cre mice.

n = 6–7 control and GCG-Gq DREADD mice were fed HCD (Teklad TD88137) ad libitum for 5 months prior to testing. (A) CNO stimulation reduced food intake following an 18-hour fast compared with control CNO-injected littermates (2-way repeated measures ANOVA, significant effects of time [F_3,33 = 72.91, ****P < 0.0001] and CNO treatment [F_1,11 = 36.04, ****P < 0.0001]). (B) Prior to testing the effects of CNO injection on body weight, control and GCG-Gq DREADD mice exhibited no difference in mass (2-way repeated measures ANOVA, F_1,11 = 0.02455, P = 0.8783). (C) Regular injections of CNO spaced 12 hours apart significantly reduced body weight in the GCG-Gq DREADD animals but had no effect on controls (2-way repeated measures ANOVA, significant effects of time [F_4,44 = 5.02, **P = 0.002] and CNO [F_1,11 = 14.59, **P = 0.0028]). (D) Fat mass was reduced (t test, t = 3.76, **P = 0.0032), while no change in lean mass (t test, P = 0.11) was seen. (E and F) No effect of CNO injection on glucose homeostasis was observed. (E) Two-way repeated measures ANOVA, significant effect of time (F_4,44 = 42.82, P < 0.0001), no effect of CNO treatment (F_1,11 = 4.182, P = 0.655). (F) Two-way repeated measures ANOVA, significant effect of time (F_4,44 = 86.73, P < 0.0001), no effect of CNO treatment (F_1,11 =4.447, P = 0.0587). No effect of CNO was observed on fed insulin levels (G, t test t = 0.758, P = 0.464) or on glucose uptake (H, n = 3 mice per group, t test for each tissue sourced from CNO-treated WT and GCG-Gq DREADD animals, EDL t = 0.058, P = 0.958; TA t = 1.604, P = 0.1840; soleus t = 0.2589, P = 0.8085; WAT t = 0.8964, P = 0.4207; liver t = 1.200, P = 0.2964).