Glucose-sensing glucagon-like peptide-1 receptor neurons in the dorsomedial hypothalamus regulate glucose metabolism

Glucagon-like peptide-1 (GLP-1) regulates energy homeostasis via activation of the GLP-1 receptors (GLP-1Rs) in the central nervous system. However, the mechanism by which the central GLP-1 signal controls blood glucose levels, especially in different nutrient states, remains unclear. Here, we defined a population of glucose-sensing GLP-1R neurons in the dorsomedial hypothalamic nucleus (DMH), by which endogenous GLP-1 decreases glucose levels via the cross-talk between the hypothalamus and pancreas. Specifically, we illustrated the sufficiency and necessity of DMHGLP-1R in glucose regulation. The activation of the DMHGLP-1R neurons is mediated by a cAMP-PKA–dependent inhibition of a delayed rectifier potassium current. We also dissected a descending control of DMHGLP-1R –dorsal motor nucleus of the vagus nerve (DMV)–pancreas activity that can regulate glucose levels by increasing insulin release. Thus, our results illustrate how central GLP-1 action in the DMH can induce a nutrient state–dependent reduction in blood glucose level.


INTRODUCTION
Glucagon-like peptide-1 (GLP-1), a gut-brain hormone, is a posttranslational cleavage product of preproglucagon encoded by the Gcg gene (1)(2)(3)(4)(5). In the periphery, GLP-1 is secreted postprandially from the gut and augments insulin release depending on the availability of glucose (3,4,(6)(7)(8)(9). In the central nervous system, GLP-1 is mainly produced by a subpopulation of neurons in the nucleus tractus solitarius (NTS) (10,11). GLP-1 neurons send robust projections to various forebrain regions, especially to the feeding control centers of the paraventricular nucleus of hypothalamus (PVN) and the dorsomedial hypothalamic nucleus (DMH) (12,13). The roles of hypothalamic GLP-1 in the regulation of feeding behavior have been intensively investigated, yet the function of central GLP-1 in the regulation of glucose metabolism remains controversial. While most studies reported a hypoglycemic effect (11,14), others have observed a hyperglycemic effect after central GLP-1 treatment (15). Thus, it is important to understand whether and how central GLP-1 affects glucose metabolism.
GLP-1 receptor (GLP-1R) is a G protein (heterotrimeric GTPbinding protein)-coupled receptor that is expressed abundantly in both the periphery (16) and the brain, including the PVN, DMH, and the arcuate nucleus (ARC) (12). Previously, we reported an intracellular signaling pathway responsible for regulating feeding behavior in the PVN, involving phosphorylation of glutamatergic AMPA receptor subunit GluA1, followed by enhanced AMPA receptor membrane trafficking postsynaptic excitation (17). However, it is not clear whether the same intracellular signaling pathway or a different one regulates glucose metabolism.
One important advantage of GLP-1-based therapies for diabetes is that GLP-1-induced insulin release is dependent on circulating glucose levels, making GLP-1-based therapies less likely than other insulin secretagogues to cause hypoglycemia (3,4). GLP-1 signaling in the brain with regard to feeding behavior and glucose metabolism is also likely to be affected by the nutritional status of the animal (8,18,19), a hypothesis that has yet to be fully investigated.
To address these outstanding questions, we investigated the neural basis of how central GLP-1 regulates glucose levels. We also addressed the possible intracellular signaling pathway induced by activation of GLP-1R in doing so. Last, we investigated how integrating the GLP-1 signal and nutrient state in the hypothalamus affects glucose levels.

Exogenous and endogenous GLP-1 in the DMH decreases glucose levels
To examine the effect of central GLP-1 on glucose levels, we use intracerebroventricular (ICV) injection of GLP-1R agonist. ICV administration of exendin-4 (Exn4) significantly decreased fasting blood glucose levels (Fig. 1, A and B). c-Fos-positive cell numbers, used as a neuronal activation marker, were increased in the GLP-1R neurons of PVN, ARC (fig. S1, A to C), and DMH ( Fig. 1, C and D) after Exn4 administration. The DMH, which receives brainstem GLP-1 projections and expresses GLP-1Rs (12,20), is a key part of the sympathetic control network (21). This suggests that it may play an important role in regulating glucose levels by GLP-1. To test this, we infused Exn4 into DMH through a bilateral guide cannula, which produced a significant reduction of fasting blood glucose levels ( Fig. 1, E and F).
Next, we asked whether endogenously released GLP-1 in the DMH has the same function as the exogenously applied GLP-1 agonist. To this end, we used Gcg-Cre transgenic mice (13,17) to selectively target NTS GLP-1 -DMH projections and manipulated their activities using both chemogenetic and optogenetic techniques. First, we injected adeno-associated virus (AAV)-mediated selective expression of synaptophysin-mGFP (membrane-targeted green fluorescent protein) into the NTS of Gcg-Cre transgenic mice ( Fig. 2A   and fig. S1E). We found dense clusters of GLP-1 neuronal fibers located in the DMH (Fig. 2B), suggesting that GLP-1-positive neurons send projections to this nucleus. We further confirmed the results by injecting retro-AAV-DIO-GFP into the DMH (injection site shown in fig. S1F) of Gcg-Cre transgenic mice (Fig. 2C). We found GFP-labeled GLP-1-positive cells, indicating that those cells were DMH projecting (Fig. 2D). Among those DMH-projecting GLP-1 neurons, more than 80% of them also project to PVN ( fig. S2, A and B).
Next, we asked whether NTS GLP-1 neurons form direct synaptic contacts with DMH neurons. Taking advantage of channelrhodopsin 2 (ChR2)-assisted circuit mapping (CRACM) (22), we expressed ChR2 in the NTS of Gcg-Cre transgenic mice and then performed brain slice recordings in the DMH neurons (Fig. 2E). We detected blue light-evoked excitatory postsynaptic currents (EPSCs) in 7 of 19 DMH neurons from four mice, indicating a possible direct synaptic connection between NTS GLP-1 neurons and DMH neurons (Fig. 2, F and G). AAV-DIO-hM 3 Dq-mCherry, a Cre-dependent stimulatory Designer Receptor Exclusively Activated by Designer Drugs (DREADDs), was injected into the NTS of Gcg-Cre mice to activate NTS GLP-1 neurons ( Fig. 2H and fig. S2C). Administration of the DREADD ligand clozapine N-oxide (CNO) by intraperitoneal injection significantly increased c-Fos-positive cell numbers in the DMH ( Fig. 2I and fig. S2D), suggesting that DMH neuronal activity can be driven by activation of NTS GLP-1 neurons.
Then, we tested whether targeted activation of NTS GLP-1 -to-DMH projections affects blood glucose level by injecting AAV-DIO-ChR2-YFP (yellow fluorescent protein) into the NTS region of Gcg-Cre transgenic mice, into which optical fibers had been implanted (bilaterally) in the DMH region (Fig. 2J). Optical stimulation of GLP-1 nerve terminals in the DMH caused a significant reduction of blood glucose in fasting animals (Fig. 2K). This result supports that optogenetic activation of NTS GLP-1 -to-DMH projection functions directly in decreasing fasting blood glucose levels. However, it will cause co-releasing GLP-1 and glutamate from activating NTS GLP-1 -to-DMH neurons (Fig. 2F). We then investigated whether the endogenously released GLP-1 is required for the control of glucose levels. To do so, we injected retro-AAV-DIO-hM 3 Dq into the DMH of Gcg-Cre mice to allow expression of excitatory DREADD-hM 3 Dq in the DMH-projecting GLP-1 neurons. By local infusion of Exn9 (a specific GLP-1R antagonist) through a guide cannula, we were able to block the GLP-1 signal in the DMH while selectively activating NTS GLP-1 -to-DMH projections ( Fig. 2L and  fig. S2E). Consistent with our optogenetic results (Fig. 2K), stimulation of NTS GLP-1 -to-DMH-projecting neurons by CNO alone decreased fasting blood glucose levels, while CNO plus DMH Exn9 treatment completely blocked the hypoglycemic effect (Fig. 2M). To examine whether chemogenetic stimulation of NTS GLP-1 neurons can produce GLP-1 release in the DMH, we collected DMH brain slice and performed enzyme-linked immunosorbent assay (ELISA) after CNO injection and found an increasing trend of DMH GLP-1 concentration when activating NTS GLP-1 neurons chemogenetically ( fig. S2F). Together, these findings suggest that endogenous GLP-1 release, induced by stimulation of NTS GLP-1 -to-DMH projections, is required for down-regulating blood glucose levels.

DMH GLP-1R is required for down-regulating blood glucose levels
Given that the presence of GLP in the DMH produced a reduction in blood glucose levels, we next asked whether this down-regulation    3 Dq-specific agonist, CNO significantly decreased fasting blood glucose when compared to control group (retro-AAV-DIO-GFP-infected Gcg-Cre mice). Again, a slight increase on vehicle group was observed, which can be induced by the experimental process. *P < 0.05, **P < 0.01, and ***P < 0.001 (ANOVA test). Pretreatment with the GLP-1R antagonist Exn9 completely blocked the NTS GLP-1 -DMH-induced decreases on fasting blood glucose level. # P < 0.05 (ANOVA test), hM 3 Dq + DMH phosphate-buffered saline (PBS) versus hM 3 Dq + DMH Exn9. The number of animal used is indicated in each panel. The data presenting style for (E) to (G) and (J) were adapted from our previous publication (17). postprandial glucose, the animals were fasted for 12 hours (overnight). Glucose levels were measured right after 1-hour refeeding, t = 0 and then t = 30 and 60 min. Food intake within 1 hour was not different between the two groups ( fig. S3K). Ablation of GLP-1R in DMH impaired postprandial glucose recovery (repeated measures, P < 0.05; Fig. 3E and fig. S3L). Consistently, administration of GLP-1R antagonist Exn9 in the DMH of wild-type (WT) animals caused a higher postprandial glucose level than was seen in the vehicle group (repeated measures, P < 0.05; Fig. 3F and fig. S3, M and N), indicating that the DMH GLP-1 signal is required for postprandial glucose recovery. Next, we asked whether GLP-1R inactivation in the DMH affects the glucoregulatory effect of peripheral liraglutide treatment, a GLP-1R agonist. As expected, fasting blood glucose levels decreased after liraglutide injection in both groups (P < 0.001, time effect, repeat measurement; Fig. 3, G and H). However, we found a different glucoregulatory effect between the two groups in response to liraglutide treatment (P < 0.001, group effect, repeat measurement), and post hoc analysis of variance (ANOVA) analysis showed that blood glucose is higher in the GLP-1R depletion group at t = 0, 30, 60, and 90 min (P < 0.001; Fig. 3F). Together, these data suggest that impaired DMH GLP-1R signaling causes dysfunction of glucose homeostasis in adult mice.

GLP-1R activation in DMH antagonizes voltage-gated delayed rectifier potassium current
The above results showed that expression of GLP-1R in the DMH is required for the reduction of blood glucose levels. We next asked whether direct activation of GLP-1R-expressing neurons in the DMH would affect glucose metabolism. The excitatory DREADD hM 3 Dq (AAV-DIO-mCherry-hM 3 Dq) was expressed in the DMH of GLP-1R-Cre mice ( Fig. 4A and fig. S4, A to C). We observed a significant decrease in fasting blood glucose level after systemic injection of hM 3 Dq-specific agonist CNO (Fig. 4B), indicating that activation of DMH GLP-1R neurons did down-regulate glucose levels.
We showed that the presence of GLP-1 or activation of GLP-1R neurons in DMH could reduce blood glucose levels. However, whether GLP-1 can activate GLP-1R neurons in DMH remains unknown. To address this question, we injected AAV-DIO-mCherry Exn4 (10 nM) to label GLP-1R neurons and characterized spontaneous action potential firing properties with current-clamp recording. Action potential firing rate showed no change with the application of Exn4 ( fig. S5, A and B), while membrane potential was significantly depolarized in GLP-1R neurons (Fig. 4, C to E), suggesting that GLP-1 can activate GLP-1R neurons in DMH. We suspected that GLP-1 could induce GLP-1R neuronal activity by affecting the synaptic transmission or ion channel opening. Application of Exn4 did not change either miniature EPSCs or miniature inhibitory postsynaptic currents ( fig. S4, E to J), suggesting that GLP-1 does not affect synaptic transmission in DMH GLP-1R neurons. Analysis of sodium and potassium channel kinetics revealed that the potassium currents were partially blocked by Exn4 ( fig. S5, C to F), suggesting that GLP-1 can activate GLP-1R neuronal activity by targeting potassium channels.
Given that the voltage-dependent potassium channel (K v ) currents include the fast A-type potassium current (I A ) and delayed rectifier potassium current (I Kd ), we recorded I Kd and I A using a method that allows for the identification of each current ( Fig. 4F and fig. S5G). To subtract I A , 50 ms of depolarized prepulses (+10 mV) was programmed before voltage steps, and I A density was subtracted from the trace of I Kd . The plot of mean currents showed that I Kd , but not I A , was significantly inhibited by application of Exn4 (repeat measurement, voltage × treatment effect, P < 0.05 for I Kd and P = 0.2 for I A ; Fig. 4G and fig. S5H). Post hoc analysis showed that the current density of I Kd at +20 mV was 116 ± 11 pA/pF in the vehicle control and 101 ± 9 pA/pF with Exn4 treatment (P = 0.01); at +30 mV, it was 155 ± 15 pA/pF in control and 133 ± 12 pA/pF with Exn4 treatment (P < 0.01); and at +40 mV, it was 196 ± 18 pA/pF in the control and 165 ± 14 pA/pF with Exn4 treatment (P = 0.001). Together, activation of DMH GLP-1R partially blocked delayed rectifier K v channels, but did not rapidly inactivate I A , resulting in suppression of outward potassium currents and depolarization.
If GLP-1 regulates DMH neuronal activity by suppressing I Kd currents, we would expect a hypoglycemic effect in response blocking K v channels on GLP-1R neurons. Consistent with this hypothesis, direct infusion of JNJ-303, a specific delayed rectifier K v blocker, into the DMH recapitulated the Exn4-induced decrease in fasting blood glucose level (Fig. 4, H and I).
Previous studies have indicated that adenosine 3′,5′-monophosphate (cAMP) may regulate I Kd (23) and that GLP-1 can activate protein kinase A (PKA) signaling (24). Therefore, we next tested whether down-regulation of I Kd in DMH GLP-1R neurons is cAMP-PKA pathway dependent. We used Rp-cAMP (a cAMP-PKA-specific blocker) during I Kd recordings. As expected, the GLP-1R activationinduced inhibition of I Kd current density was completely blocked by Rp-cAMP (repeat measurement, voltage × treatment effect, P = 0.542; Fig. 4, J and K), indicating that activation of the cAMP-PKA pathway is required for the effect of GLP-1 on I Kd .
Following the discovery that Rp-cAMP can block the GLP-1induced inhibition of I Kd current density, we next asked whether Rp-cAMP can block the GLP-1-induced suppression of blood glucose levels. To test this, we infused Rp-cAMP into the DMH before application of Exn4 through a guide cannula. We found that cotreatment with Rp-cAMP blocked Exn4-induced down-regulation of fasting blood glucose level (group effect, vehicle versus Exn4 treatment, P < 0.01; vehicle versus Exn4 + Rp-cAMP treatment, P = 0.752; Fig. 1F), suggesting that activation of the cAMP-PKA pathway in the DMH is required for GLP-1's effect on blood glucose levels.

Nutritional status regulates the decreasing effect of GLP-1 on blood glucose levels
Although activation of DMH GLP-1R neurons can decrease blood glucose levels in both fasting and refed mice (Figs. 4, A and B, and 5, A and B, and fig. S4D), previous studies indicate that central GLP-1 regulation of feeding behavior is likely dependent on the nutritional status (8,18). It is still unknown whether DMH GLP-1's effect on glucose metabolism is affected by nutrient states. To address this question, we intraperitoneally injected glucose (2 g/kg body weight) to mimic a high nutrient state in mice. When administrating GLP-1R agonist Exn4 to DMH through a bilateral guide cannula, we found ±52% down-regulation of blood glucose level under high nutrient state (with glucose injection) versus ±19% under low one (without glucose injection) (Fig. 5, C and D). This indicated that Exn4 has a higher ability to decrease blood glucose levels under a high nutrient state than a low one. One of the possible reasons is that Exn4 has a different effect on GLP-1R neuronal activities under different nutrient states.
To understand the GLP-1's effect on GLP-1R neuronal activities under different nutrient states, we use 1 and 5 mM glucose artificial cerebrospinal fluid (ACSF) to mimic the low and high nutrient states, respectively, in vitro. We labeled GLP-1R neurons of the DMH by injecting AAV-DIO-mCherry virus in GLP-1R-Cre mice and performed current-clamp recordings following either (i) vehicle perfusion (1 mM glucose, ACSF); (ii) Exn4 + 1 mM glucose, ACSF; or (iii) Exn4 + 5 mM glucose (Fig. 5E). When compared to vehicle, both Exn4 + 1 mM glucose and Exn4 + 5 mM glucose groups showed depolarized membrane potential (Fig. 5, F and G). A significantly increased action potential firing rate was observed in the Exn4 + 5 mM glucose group, but not in the Exn4 + 1 mM glucose group (Fig. 5, F and H). It suggests that GLP-1's stimulating effect on GLP-1R neurons becomes more robust when higher glucose is presented, further supporting a synergistic effect between GLP-1 and glucose.
One possible reason to induce the synergistic effect between GLP-1 and glucose is that DMH GLP-1R neurons are glucose sensing. To this end, we investigated whether 5 mM glucose alone can activate DMH GLP-1R neurons. AAV-DIO-GCaMP6 virus was injected into GLP-1R-Cre mice to allow GCaMP6, a calcium signal indicator, expressed in the DMH. Coronal hypothalamic slices (containing DMH) were prepared the same way as an electrophysiological experiment. When switching the perfusion solution from 1 to 5 mM, 20 of 29 cells (from four mice) showed a significant change in F/F. Among the 20 cells, 19 of them showed increased activity during 5 mM glucose perfusion, while one cell showed decreased activity, suggesting that most GLP-1R neurons in the DMH are excited by 5 mM glucose (Fig. 5, I and J). Next, we recorded the neuronal firing activity of GLP-1R neurons in response to 5 mM glucose using whole-cell patch-clamp recording in current-clamp mode. Fifteen of 23 GLP-1R-positive neurons (from four mice) showed at least a 20% change in membrane potential, with 13 cells showing a depolarized potential; two others showed hyperpolarization (Fig. 5, K and L). Together, our experiments have identified a population of glucose-sensing GLP-1R neurons within the DMH.
We also conducted in vivo experiments wherein AAV-DIO-GCaMP6 was injected into DMH of the GLP-1R-Cre mice, and an optic fiber was subsequently implanted in the same position to monitor calcium signal changes ( fig. S6, A and B). To mimic a high nutrient state, animals were trained to drink mixed glucose (10%) 7 of 14 and sucrose (2.5%) water solution after overnight food and water deprivation. As expected, blood glucose increased in 5 min of glucose consumption (10% glucose + 2.5% sucrose, ±11.7 mg/dl versus tap water ±6.5 mg/dl; fig. S6, C and D). Concurrently, the Ca 2+ signal increased (indicated by F/F) by about 30% compared to baseline. No significant changes in bulk Ca 2+ signals were found when animals consumed tap water (Fig. 5, M and N), suggesting that DMH GLP-1R neurons can sense circulating glucose levels.
Descending control of DMH-DMV-pancreas activity mediates the decreasing effect of GLP-1 on blood glucose levels Next, we sought to identify downstream targets of DMH GLP-1R neurons that might mediate glucose-lowering effects. Following injection of AAV-DIO-synaptophysin-GFP into the DMH of GLP-1R-Cre mice ( Fig. 6A and fig. S6E), dense labeled terminals were found in brainstem regions, including the NTS, dorsal motor nucleus of the vagus nerve (DMV), and raphe pallidus nucleus (RPa), but not in the

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intermediolateral nucleus (IML; Fig. 6B and fig. S6, F and G). Although both dorsal vagal complex (DVC) and RPa received afferents from DMH GLP-1R , chemogenetic stimulation of DMH GLP-1R increased c-Fos expression in the DVC, but not in RPa ( fig. S6, H to J). Because DMV plays a critical role in mediating peripheral organ activity, such as liver and pancreas, we further examined these projections using output-specific monosynaptic viral tracing (25). GLP-1R-Cre mice were infected with AAV-DIO-TVA-dsRed and AAV-DIO-RV-G in the DMH and RV-EnvA-G-GFP in the DMV 4 weeks later (Fig. 6C). Consistent with ChR2-mediated tracing data, frequent GFP + cell bodies were found in the NTS (Fig. 6D), suggesting that an NTS-DMH GLP-1R -DMV circuit can be involved in regulating glucose metabolism.
To answer whether the DMH GLP-1R -DMV circuit regulates glucose levels, we injected AAV-DIO-ChR2-YFP into the DMH region of GLP-1R-Cre mice, into which optical fibers had been implanted in the DMV region ( Fig. 6G and fig. S6, K and L). Optical stimulation of GLP-1R nerve terminals in the DMV caused a significant reduction of blood glucose in fasting animals (Fig. 6H), indicating that stimulation of the DMH GLP-1R -DMV circuit down-regulates glucose levels.
DMV sends the descending fibers of the vagus nerve to both liver and pancreas branches, which regulates glucose homeostasis and insulin release, respectively (26,27). To isolate the "brain-periphery" connection between DMH GLP-1R neurons and peripheral metabolic center, we injected AAV-DIO-mCherry in the DMH of GLP-1R-Cre  mice and the trans-synaptic retrograde tracing agent, pseudorabies virus (PRV), into the liver and pancreas, respectively ( Fig. 6E and  fig. S6M). We found frequent GFP + neurons in DMH when injecting PRV-GFP into the pancreas, while few GFP + neurons were found when injecting PRV-GFP into the liver (Fig. 6F and fig. S6N), suggesting a DMH-pancreas connection instead of DMH-liver connection. We found colocalization of GFP-labeled pancreas PRV and mCherry-labeled GLP-1R in the DMH, indicating a DMH GLP-1Rpancreas projection (Fig. 6F). Thus, stimulation of DMH GLP-1R may regulate pancreas activity, resulting in insulin level change. To test this, we infused Exn4 into DMH via bilateral guide cannulas and found that plasma insulin levels significantly increased when compared to the vehicle group (Fig. 6, I and J). Consistently, stimulation of DMH GLP-1R neuronal activity chemogenetically also upregulated plasma insulin levels (Fig. 6, K and L), while inactivating GLP-1R in DMH decreased plasma insulin levels (Fig. 6, M and N). It suggests that DMH GLP-1's effect on glucose levels can be induced by insulin release through the cross-talk between the hypothalamus and pancreas.

DISCUSSION
In the present study, we discovered a population of glucose-sensitive GLP-1R neurons in the DMH, by which endogenous GLP-1 downregulates glucose level via blockage of delay rectifier potassium channels in a cAMP-PKA-dependent pathway. The input-output organizations revealed that the activation of DMH GLP-1R -induced glucose-lowering effect can be mediated by descending controls of hypothalamus-brainstem-pancreas activity.
Although previous studies suggest that peripheral, rather than central, GLP-1R signaling is essential for the glucose-lowering effect mediated by this receptor (28,29), acute manipulation of central GLP-1 signaling has also been shown to regulate glucose homeostasis (14,28,30). NTS GLP-1 neurons receive direct synaptic inputs from vagal afferent neurons (31,32). It has been reported that gastrointestinal distension and large liquid intakes can induce c-Fos expression in this NTS population (33,34). Recent studies indicated that NTS GLP-1 projecting vagal afferent neurons could be activated by peripheral oxytocin but not GLP-1 (35). It is a fundamental question of how the ascending information is sent from NTS GLP-1 to the hypothalamus and processed to control energy metabolism. In a previous study, we discovered an NTS GLP-1 -PVN pathway, by which central GLP-1 can regulate feeding behavior (17). Here, we presented an NTS GLP-1 -DMH pathway down-regulating blood glucose level, suggesting that the DMH GLP-1 signal is able to regulate glucose homeostasis. Both PVN and DMH receive ascending afferents from NTS GLP-1 (fig. S2, A and B). Although PVN-projecting and DMH-projecting GLP-1 neurons are largely overlapped, functions mediated by these two nuclei are different. As the PVN GLP-1 functioned on feeding behavior (17,28), DMH GLP-1 altered glucose levels and energy metabolism. The different readout can be induced by targeting glucose-sensing GLP-1R neurons in the DMH and the downstream vagal controls.
Sensing of peripheral glucose levels by the hypothalamus plays an important role in maintaining blood glucose levels. Glucosesensing neurons have been identified in several hypothalamic nuclei: ARC, lateral hypothalamus, paraventricular nucleus, and ventromedial nucleus (36)(37)(38)(39)(40). It has also been reported that glucose-sensing neurons are found in the DMH (41). Glucose-sensing neurons are broadly divided into two groups: glucose-excited (GE) and glucoseinhibited (GI). GE neurons show enhanced neuronal activity in response to higher ambient glucose concentrations, while the reverse is true for GI neurons (36,(42)(43)(44). Our results revealed that most glucose-sensing GLP-1R neurons are the GE type, by which central GLP-1 signaling and peripheral glucose signals are integrated to regulate glucose levels.
GLP-1 regulation of feeding behavior and glucose level is known to be dependent on the nutritional state (8,18). Here, we used 1 and 5 mM glucose to mimic low and high nutrient states, respectively (38,45). Although either GLP-1R agonist Exn4 or 5 mM glucose can activate GLP-1R neurons to a certain degree, the activation effect became more robust when both Exn4 and 5 mM glucose were present. This indicates that Exn4 can have a synergistic effect with glucose on the regulation of GLP-1R neuronal activity. Thus, we hypothesize that, as a high nutrient state, the DMH GLP-1 signal affects postprandial glucose recovery. Our GLP-1R knockout model and Exn9 antagonist experiments suggest that the DMH GLP-1R signal can be required for postprandial glucose recovery.
Using rigorous viral-mediated neural tracing, we can dissect the connections between central GLP-1R neurons and the peripheral metabolic center, i.e., the pancreas. Our results support that DMH GLP-1R neurons control pancreas rather than liver activity. Neural-pancreatic research has revealed that, in addition to the pancreatic hormones and peptides, the DVC is a major brain region involved in mediating insulin secretion (46,47). In our study, the DMH GLP-1R -DMV circuit regulates glucose levels. Thus, we demonstrated a descending control of DMH GLP-1R -DMV-pancreatic activity, mediating the central GLP-1 effect on glucose metabolism (Fig. 7A), although RPa, which mediates a sympathetic activity (41,48), also receives some afferents from DMH GLP-1R (fig. S6G). There are only a few c-Fos + cells in the RPa when compared to the DVC after stimulating DMH GLP-1R neurons. It suggested that DMH GLP-1R neurons primarily targeted parasympathetic nerve to control pancreas activity. Our results are consistent with previous studies, which indicated that DMH neurons are the secondorder parasympathetic pancreas-projecting neurons or could be the third-order sympathetic pancreas-projecting neurons (49). In addition, a close interaction between the parasympathetic and sympathetic motor nuclei may also regulate insulin release (50,51). Further studies are needed to address whether a dual regulatory role is induced by DMH GLP-1R on both sympathetic and parasympathetic nerve activity.
Although recent studies showed that conditional knockout of GLP-1R in nestin-Cre or Nkx2.1-Cre mice had no impact on the glucose metabolism (28,29), we suspect that, in this instance, a developmental compensatory effect can occur during embryogenesis. In the present study, we used strategies involving the injection of AAV-Cre to block GLP-1R expression in adult GLP-1R f/f mice. As our data demonstrated, impaired GLP-1R expression in the DMH caused an increased blood glucose level without interrupting food intake, which is different from what we observed in PVN (17), indicating that a distinct GLP-1 effect can exist between PVN and DMH. There was a glucose surge at around 4 weeks after virus injection, followed by a drop to a relatively lower and stable level, implying that a compensatory mechanism for normalizing glucose levels may develop over time. The increased glucose level is not likely induced by the secondary effect of feeding because overnight feeding is not altered during the 7 weeks of observations. Although we found an increase in body weight gain at week 5, the change in glucose levels was earlier (at week 4). Likewise, the body weight gain is likely to be induced by decreased energy expenditure in GLP-1R knockdown mice. Considering that changes in energy expenditure may also affect glucose metabolism and insulin release, we cannot exclude the possible secondary glucoregulatory effect resulting from energy metabolism.
We previously showed that GLP-1R activation in the PVN augments excitatory synaptic strength in PVN neurons, with GLP-1R activation promoting a PKA-dependent signaling cascade leading to phosphorylation of serine-845 on GluA1 AMPA receptors and their trafficking to the plasma membrane (17). The present investigation of GLP-1R neuron activation in the DMH detected no such AMPA receptor-mediated downstream signaling events. Instead, our data suggest that GLP-1R activation suppresses delayed rectifier potassium channel-mediated currents, rather than rapidly inactivating potassium currents, which results in the depolarization of the membrane potential. Previous studies showed that the cAMP-PKA pathway could suppress delayed rectifier K v channels (5,23,24,(52)(53)(54)(55); our work suggests that the GLP-1R activationinduced reduction of K v can also be controlled by cAMP-PKA activity. Thus, our data indicate that the signaling pathway involved in the GLP-1 regulation of DMH neurons can progress as follows: An elevation in intracellular cAMP levels activates PKA signaling, which leads to the blockade of the delayed rectifier potassium channel, resulting in postsynaptic neuronal depolarization and excitation (Fig. 7B).
We want to point out that although we dissected a descending control from central GLP-1 to pancreas activity in this study, we cannot exclude other possible factors that may contribute to the glucoregulatory effect, i.e., the energy expenditure. Moreover, we do not have direct evidence to show that the vagal control insulin release is by targeting  cells of the pancreas islet. Further studies are needed to address those exciting questions.
In summary, our studies highlighted the importance of central GLP-1 signaling in nutrient state-dependent regulation of glucose metabolism by targeting glucose-sensing GLP-1R neurons in the DMH. The input-output organizations revealed that a descending control of DMH-brainstem-pancreas activity could contribute to the glucose-lowering effect.

Animals
All procedures involving mice were approved by the Institutional Animal Care and Use Committee from Rutgers Robert Wood Johnson Medical School and the University of Science and Technology of China. All animals used in this study were 6-to 8-week-old males unless otherwise stated. The animals are bred in the facility at ~22°C and 35 to 55% humidity. The Gcg-Cre transgene was created by insertion of Cre-recombinase into the Gcg gene locus of BAC (bacterial artificial chromosome) RP23-242F22 at the ATG start codon as previously described (13). GLP-1R-Cre mice were purchased from The Jackson Laboratory (strain no. 029283). WT mice C57BL/6 and GLP-1R-flox mice were purchased from GemPharmatech (T005818, Nanjing, China). WT mice C57BL/6 were purchased from The Jackson Laboratory. In all cases, mice were randomized according to body weight. The investigator was blinded to the treatment groups except electrophysiological experiments. The sample size required was estimated to be n = 6 to 12 per group on the basis of previous studies examining the effects of GLP-1 on feeding behavior (17). All procedures had been conducted by at least three biological repeats.

AAV virus
The AAV viruses used in this study include AAV-hSyn-DIO-hM 3 Dq-mCherry, retro-AAV-hSyn-DIO-hM 3 Dq-mCherry, AAV-hSyn-DIO-mCherry, AAV-EF1a-DIO-ChR2-YFP, retro-AAV-hSyn-DIO-GFP, AAV-hSyn-Cre, and AAV-EF1a-DIO-GFP (purchased from the University of North Carolina Gene Therapy Center). The ChR2-YFP encodes a membrane-bound fusion protein, allowing visualization of both the cell bodies and axons of Cre-expressing neurons for morphologic analyses (17). Viral-mediated protein expression was allowed for a period of 14 days before experimental manipulation. Injection sites were confirmed by cutting brain sections and inspecting them under a stereoscope or microscope in all animals reported in this study (17). In total, 0.2 l of virus was used for the injection of the Cre-dependent expression AAV virus, and 60 nl of virus was used for the injection of non-Cre-dependent expression (17). The injection speed was 0.1 l/5 min, while the injection coordinates for NTS were as follows: anteroposterior (AP), −3.4 mm from lambda; lateral (L), ±0.5 mm; ventral (V), 4.125 mm. The injection coordinates for the DMH were as follows: AP, −1.6 mm from bregma; L, ±0.5 mm; V, 5.0 mm. For PRV-mediated trans-synaptic tracing, 0.1 l of AAV-DIO-mCherry was injected in DMH of GLP-1R-Cre mice. Two weeks later, 0.25 l of PRV-GFP was injected into the liver or pancreas and then the mice were sacrificed 5 days after PRV injection. Hypothalamic slices were harvested and observed by confocal microscopy (Zeiss, LSM880).

ChR2-assisted circuit mapping
Gcg-Cre animals were injected by AAV-hSyn-DIO-ChR2-YFP into NTS. Two weeks after surgery, mice were deeply anesthetized with Euthasol before sacrifice. Coronal hypothalamic slices (300 m) were prepared, and whole-cell patch-clamp recordings were performed at 30°C in ACSF containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 25 mM NaHCO 3 , 2.5 mM glucose, 22.5 mM sucrose, 2.5 mM CaCl 2 , and 1.2 mM MgCl 2 . Photostimulationevoked EPSCs were recorded in the whole-cell voltage-clamp mode, with membrane potential clamped at −70 mV (17). Photostimulationevoked EPSCs were recorded in the presence of picrotoxin (100 M) to block inhibitory postsynaptic currents (17). All recordings were made using a multiclamp 700B amplifier, and data were filtered at 2 kHz and digitized at 10 kHz. To photostimulate ChR2-positive fibers, a light-emitting diode (LED) light source was used to generate 470-nm blue light, which was focused on the back aperture of the microscope objective, producing a wide-field exposure of the DMH area (17). The photostimulation-evoked EPSC detection protocol was used as described previously (56) and involved four blue light laser pulses administered 1 s apart during the first 4 s of an 8-s sweep, repeated for a total of 16 sweeps.

Depletion of GLP-1R expression in DMH
Each cage of GLP-1R f/f mice (four animals) was divided into two groups: Two of them were injected with the AAV-Cre virus into the DMH bilaterally using the following coordinates: AP, −1.6; L, ±0.5; V, 5.0. Another two received an injection of the control virus (AAV-GFP virus). The animals were allowed to recover from surgery for 2 weeks. Body weight, fasting glucose level, and overnight food intake were monitored every week, 2 weeks after surgery. For the refed glucose, the animals were fasted for 12 hours (overnight). The first time point (t = 0) for glucose measurement is right after 1-hour refeeding, and then glucose levels were measured at t = 30 and 60 min. At week 4, we intraperitoneally injected liraglutide (300 g/kg; Novo Nordisk) in both the DMH GLP-1R ablation group and control group. The blood glucose levels were measured at t = 0 (before drug injection) and t = 30, 60, and 90 min. The animals were sacrificed at week 7; epididymal fat was separated and measured. The brains were cut in precold ACSF by 300 m thickness, the DMH was separated immediately, mRNA was extracted for quantitative polymerase chain reaction (qPCR) analysis, and protein was saved in -80°C until further analysis. For Western blotting, anti-GLP-1R (ab39072, Abcam, RRID: AB_880213) and horseradish peroxidase anti-rabbit (31460, Life Technologies, RRID: AB_228341) were used for primary and secondary antibody, respectively. For qPCR, the primers used for GLP-1R and GAPDH gene are as follows: GLP-1R, GGGCCAG-TAGTGTGCTACAA (forward) and CTTCACACTCCGACAGGTCC (reverse); GAPDH, GTTTGTGATGGGCGTGAACC (forward) and ATGCCGAAGTTGTCGTGGAT (reverse).

Behavior experiments Optogenetic manipulation of GLP-1 fibers.
Optical fibers [200 m diameter core, numerical aperture (NA) 0.37] (Thorlabs) were coupled to a fiber optic rotary joint (Prizmatix) and an LED generator (470 nm for stimulation, Prizmatix UHP-T-470-LA) (17). A terminal fiber attached to the rotary joint was coupled to a 1.25-outside diameter (OD) zirconium ferrule and a mating sleeve that allowed the delivery of light directly to the brain (17). For in vivo photostimulation experiments, 5-ms pulses and 470-nm blue light were given at 10 pulses per second every 4 s for 1 hour (17). For the stimulation experiment, animals were provided only 0.5 g of food overnight. All the mice were trained for 4 days to acclimate to the feeding schedule. To measure the fasting blood glucose, a small drop of tail-tip blood (<5 l) was placed on the test strip of the blood glucometer (Abbott, FreeStyle Freedom Lite) (17).

DREADD experiment
We injected retro-AAV-DIO-hM 3 Dq-EGFP (enhanced GFP) into DMH in Gcg-Cre transgenic animals to enable DMH-projecting GCG neurons to express hM 3 Dq and then implanted a bilateral guide cannula onto DMH. After 3 weeks of recovery and virus expression, the mice were trained for an additional 4 days to acclimate to the feeding schedule. The animals were then divided into three groups: (i) control (EGFP) + CNO (intraperitoneal injection, 1 mg/kg) + DMH PBS; (ii) hM 3 Dq + CNO (intraperitoneal injection, 1 mg/kg) + DMH PBS; and (iii) hM 3 Dq + CNO (intraperitoneal injection, 1 mg/kg) + DMH Exn9 (10 M, 0.5 l). Fasting blood glucose was monitored at t = 0, 30, 60, and 90 min. For the refed glucose, the animals were fasted for 12 hours (overnight). The first time point (t = 0) for glucose measurement is right after 1-hour refeeding, and then glucose levels were measured at t = 30 and 60 min.

Cannula infusion
The bilateral guide cannulas (RWD, 62024) were implanted into the DMH and fixed to the skull with three screws and cement. The coordinates used for the DMH were as follows: AP, −1.6; L, ±0.5; V, 4.8. After surgery, mice were housed individually, and 2 weeks were allowed for recovery. The cannula location was checked after sacrificing of animals, and data were collected only from animals with proper placement.
To study the effect of the GLP-1 signal on postprandial glucose, the animals were fasted for 12 hours (overnight). Exn9 (0. 1 g/200 nl; Tocris, 1933) was injected into DMH immediately after 1-hour refeeding, and the first time point (t = 0) for glucose level was measured. Then, glucose levels were measured at t = 30 and 60 min.
To block the I Kd pathway in the DMH, 200 nl (2.5 M) of the I Kd inhibitor JNJ-303 (MedChem Express, HY-16953) was locally injected through the guide cannula into DMH. Fasting blood glucose was monitored at t = 0, 30, 60, 90, and 120 min.

Hormone measurement
For blood glucose measurement, a drop of tail blood was applied by glucometer (FreeStyle Lite system, Abbott Diabetes Care Inc., CA, USA). For insulin measurement, about 150 l of trunk blood was collected before scarification. The plasma samples were harvested and froze until ELISA by centrifuging the blood samples (with anticoagulant) at 1500 rpm for 15 min (4°C). ELISA was performed following the procedure provided by ELISA kits (EMINS, Thermo Fisher Scientific).
For GLP-1 measurement, we express AAV-DIO-hM 3 Dq in the NTS of Gcg-Cre mice. After intraperitoneally injecting DREADD agonist CNO, we collected the fresh DMH brain slice (300 m slice × 2) in lysis buffer and then performed ELISA (CSB-E08118, CUSABIO) according to the provided procedure with kits.

Electrophysiology
Electrophysiology was performed as described previously (57) with minor modifications. Briefly, mice were deeply anesthetized with Euthasol before sacrifice. Coronal hypothalamic slices (300 m) were prepared, and whole-cell patch-clamp recordings were performed at 30°C in ACSF containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 25 mM NaHCO 3 , 2.5 mM glucose, 22.5 mM sucrose, 2.5 mM CaCl 2 , and 1.2 mM MgCl 2 . Patch pipettes (3.5 to 6 megohms) were pulled from borosilicate glass. Exn4 (10 nM) was applied to activate GLP-1R. For all the slice electrophysiological recordings, we target the DMH region from bregma −1.4 to bregma −2.0 and collected two coronal slices (300 m each slice). We randomly targeted GLP-1R cells in the dorsal and ventral part of DMH for recording (as we found that only few GLP-1R-positive cells present in the DMH core region; fig. S4B).
For the voltage-clamp protocol, the internal solution contains 126 mM K-gluconate, 10 mM Hepes, 4 mM KCl, 1.7 mM MgCl 2 , 4 mM Mg-adenosine triphosphate (ATP), 0.3 mM Na 4 -guanosine triphosphate (GTP), and 10 mM phosphocreatine. For the I-clamp protocol, the internal solution contains 126 mM K-gluconate, 4 mM KCl, 10 mM Hepes, 4 mM ATP-Mg, 0.3 mM GTP-Na 2 , and 10 mM phosphocreatine. GLP-1R neurons were labeled by the previous injection of AAV-DIO-mCherry into the DMH of GLP-1R-Cre mice. Whole-cell patch-clamp recordings were performed using an Axon 700B amplifier. Data were filtered at 2 kHz, digitized at 10 kHz, and collected using Clampex 10.2 (Molecular Devices). To record EPSCs, picrotoxin (50 M; Sigma-Aldrich) was added to block miniature inhibitory postsynaptic currents mediated by -aminobutyric acid type A (GABA A ) receptors. Tetrodotoxin (1 M) was added to block action potentials for mEPSC recording. To block the cAMP-PKA pathway, 50-m cAMP-Rp was applied to ACSF together with 10 nM Exn4. To block I Kd , 250 nM JNJ-303 was applied to ACSF together with 10 nM Exn4.
For recording brain slices under different energy states, mice were fasted overnight (12 hours). Fresh brain slices were made and recovered in low-glucose (1 mM) ACSF solution. During I-clamp recording, 1 mM glucose ACSF was perfused for 5 min to record baseline action potential, followed by another 5 min of 10 nM Exn4 + 1 mM glucose ACSF perfusion, and then 10 nM Exn4 + 5 mM glucose perfusion.
For testing glucose-sensing neurons, mice were fasted overnight (12 hours). The same procedure was used as described above to collect brain slices. During I-clamp recording, 1 mM glucose ACSF was perfused for 5 min to record baseline action potentials, followed by 5 mM glucose perfusion.
Ca 2+ image and fiber photometry AAV-hSyn-DIO-Gcamp6 (200 nl) was unilaterally injected in DMH of GLP-1R-Cre mice and allowed to express for 3 weeks. The animals were fasted for 12 hours before the experiment.
For in vitro experiments, animals were sacrificed in the morning, and fresh DMH brain slices were made as described in the electrophysiology section. Ca 2+ images were collected at low-glucose condition (1 mM) and high-glucose condition (5 mM), respectively, by 10 frames/s at a resolution of 1024 m × 1024 m (Hamamatsu, C11440). The data were summarized by ImageJ and MATLAB software.
For in vivo experiments, optical fibers (250 m diameter core, NA 0.37) (Newdoon) were placed on DMH and connected to a fiber photometry setup (ThinkerTech, Nanjing, China). The emission light was generated by a 480-nm LED, reflected with a dichroic mirror, and delivered to the brain to excite the GCaMP6. The emission light was passed through another band-pass filter, into a CMOS (complementary metal-oxide semiconductor) detector (Thorlabs Inc.; DCC3240M), and finally recorded by a LabVIEW program (TDMSViewer, ThinkerTech, Nanjing, China). The animals were trained for 4 days to acclimate to the experiment schedule: (i) water and food deprivation (12 hours), (ii) 30-min baseline recording, and (iii) place water bottle containing either vehicle (tap water) or glucose (tap water + 10% glucose + 2.5% sucrose).
Fiber photometry signals were processed with a custom-written MATLAB software, which is available at https://zenodo.org/ record/6456623#.YlaF_8hByUk (Zenodo ID: 6456623; Zenodo DOI: 10.5281/zenodo.6456623; ThinkerTech Nanjing Bioscience, 2020). Briefly, all the data were segmented on the basis of the behavioral events and baseline phase. The change in fluorescence (F/F) was calculated as (F − F 0 )/F 0 , where F 0 represents the baseline fluorescence signal averaged over a 10-s-long control time window.

Quantification and statistical analysis
Statistical analysis was performed using SPSS (version 18) or Excel (version 2013). We first determined whether the data values came from a normal distribution by Kolmogorov-Smirnov test. Repeatedmeasures two-way ANOVA and post hoc Bonferroni tests were used for all time-dependent experiments (17). The cumulative frequency curve was analyzed by the Kolmogorov-Smirnov test (17). Outliers are removed by using "Descriptive Statistics" of SPSS. Statistical significance was set at P < 0.05. All the details of the experiments can be found in the figure legends. All data values are presented as means ± SEM.

SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at https://science.org/doi/10.1126/ sciadv.abn5345 View/request a protocol for this paper from Bio-protocol.