hugin+ neurons were a novel glucose sensor in the fly brain.

(A) Experimental procedure schematic. Newly eclosed flies were gathered and provided with regular food for 5 days, followed by a 24-hour period of starvation. Proboscis Extension Response (PER) assays were conducted. (B) Fraction of flies showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). (C-D) Fraction of flies of the indicated genotypes under 30 °C showing PER to 400 mM of sucrose (n=4-5 groups, each of 10 flies). (E) Fraction of indicated flies showing PER to different concentrations of sucrose (ND represent standard fly food) (n=4-5 groups, each of 10 flies). (F) hugin expression in the brain, illustrated by mCD8::GFP expression driven by huginGAL4. Scale bar represents 100 μm. (G) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons during the perfusion of glucose with or without TTX (n=6-7). Horizontal black bar represents the duration indicated glucose solution stimulation. (H) AstA expression in the brain, illustrated by mCD8::GFP expression driven by AstAGAL4. Scale bar represents 100 μm. (I) Representative traces and quantification of ex vivo calcium responses of AstA+ neurons during the perfusion of glucose with or without TTX (n=6). (J) Representative images of pre-photoconversion (pre-PC) and post-photoconversion (post-PC) CaMPARI signal in hugin-expressing neurons (upper). The Red:Green ratio represents intracellular Ca2+ concentrations (lower). Scale bar represents 100 μm (n=18-23). ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Student’s t-test, one-way and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

Glucose activated hugin+ neurons via Gltu1 and KATP channel.

(A) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons during the perfusion of glucose with or without phlorizin (1mM, n=6-7). Horizontal black bar represents the duration indicated glucose solution stimulation. (B) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons in indicated flies during the perfusion of glucose. (C) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). (D-E) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons during the perfusion of glibenclamide (100 μM, D), glucose with alloxan (10 μM, E). (F) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons in indicated flies during the perfusion of glucose. (G) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons during the perfusion of pyruvate (50 mM, n=6). (H) Representative traces and quantification of ex vivo calcium responses of hugin+ neurons during the perfusion of D-glucose and L-glucose (50 mM, n=6) ns, P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001. Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

hugin+ neurons activated AstA+ neurons through PK2-R1.

(A) Fraction of flies showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). Flies were injected with saline or synthetic hugin for 30 minutes before the assay. (B) Representative traces (upper) and quantification (lower) of peak calcium transients of Gr5a+ neurons in indicated flies upon 5% sucrose after injection of synthetic hugin (n=6). Horizontal black bar represents the duration sucrose stimulation. (C-D) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). (E) Co-localization (dashed box) of PK2-R1+ neurons (green) and AstA+ neurons (red) in the SEZ region. Scale bar represents 100 μm. (F) Representative traces (upper) and quantification (lower) of peak calcium transients of AstA+ neurons after the photo-activation of hugin+ neurons (n=6) from in vivo calcium imaging. Horizontal black bar represents the duration of red-light stimulation. (G-H) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

AstA+ neurons inhibited Gr5a+ neurons through AstA-R1.

(A) Fraction of flies showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). Flies were injected with saline or synthetic AstA for 30 minutes before the assay. (B) Representative traces (left) and quantification (right) of peak calcium transients of Gr5a+ neurons in indicated flies upon 5% sucrose after injection of synthetic AstA (n=6). Horizontal black bar represents the duration sucrose stimulation. (C) Localization of Gr5a+ neurons (green) and AstA+ neurons (red) in the brain. Scale bar represents 100 μm. (D) Co-localization of Gr5a+ neurons (green) and AstA-R1+ neurons (red). Arrows point to the neurons co-expressing the two receptors. Scale bar represents 50 μm. (E-F) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). (G-H) Representative traces (left) and quantification (right) of peak calcium transients of Gr5a+ neurons in indicated flies upon 5% sucrose during the process of photo-activation of AstA+ neurons (G, n=6) and hugin+ neurons (H, n=7-8). Shadows represents the duration of red-light stimulation. Horizontal black bar represents the duration of sucrose stimulation. ns, P > 0.05; *P < 0.05; ***P < 0.001; ****P < 0.0001. Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

NMU+ neurons were the central energy sensor in mouse.

(A) Blood NMU levels of starved mice re-fed with 20% sucrose (n=6). (B) Two-bottle preference tests for starved mice re-fed with sucrose (n=7). (C) Two-bottle preference tests for starved mice with or without intraperitoneal injection of NMU peptide (YFLFRPRN-NH2, 4.5μm/kg) (n=7). (D) Two-bottle preference tests for indicated starved mice. (E) Fiber photometry to record the calcium dynamics of NMU+ neurons in the VMH with GCaMP6m. (F-G) Representative trace (left) and heatmaps (right, n=5) showing calcium dynamics of NMU+ neurons in the VMH in response to glucose perfusion. (H) Experimental approach to assess calcium signaling in NMU+ neurons in the VMH in vitro. (I-J) Representative traces and quantification of ex vivo calcium responses of NMU+ neurons during the perfusion of glucose with or without TTX (I), Alloxan (J) and Phlorizin (J) (n=6-7). Horizontal black bar represents the duration indicated glucose solution stimulation. (K) Anterograde tracing the downstream terget of NMU neurons. (L) Fiber photometry to record the calcium dynamics of Calb2+ neurons in the rNST with GCaMP6m. (M) Representative traces and quantification of calcium responses of Calb2+ neurons during glucose feeding with or without NMU injection. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Student’s t-test and one-way ANOVA followed by post hoc test with Bonferroni correction was used for multiple comparisons when applicable.

Satiety suppressed sweet sensitivity in a dopamine-independent manner.

(A) Representative traces (left) and quantification (right) of peak calcium transients of Gr5a+ neurons in indicated flies (n=6). Horizontal black bar represents the duration 5% sucrose stimulation. (B-D) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (B-C, n=4 groups, each of 10 flies). The Area Under the Curve (AUC, D) represents sweet sensitivity in C. *P < 0.05; ****P < 0.0001. Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

The effect of satiety signals on taste modulation.

(A) Fraction of fed flies of the indicated genotypes and environmental temperatures showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). (B) Fraction of starved flies of the indicated genotypes and environmental temperatures showing PER to different concentrations of sucrose (n=4-5 groups, each of 10 flies). ns, P > 0.05; *P < 0.05; ***P < 0.001; ****P < 0.0001. Two-way ANOVA followed by post hoc test with Bonferroni correction was used for multiple comparisons when applicable.

Activation of IPCs decreased the circulating sugar levels and increased sweet sensitivity.

(A) Hemolymph glucose levels from the indicated genotypes and treatment temperatures (n=8-9, 1 hour at 30 °C). (B-C) Fraction of fed flies of the indicated genotypes and environmental temperatures showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). ns, P > 0.05; ****P < 0.0001. Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

Sugar intake suppressed sweet sensation.

(A) Fraction of indicated flies showing PER to different concentrations of fructose (n=4 groups, each of 10 flies). (B) Fraction of indicated flies showing PER to different concentrations of trehalose (n=4 groups, each of 10 flies).****P < 0.0001. two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

Starvation led to a decrease in the circulating sugar levels.

Hemolymph glucose levels from indicated flies (n=6-8). ***P < 0.001. Student’s t-test was used for comparisons.

The response of different neuropeptide-expressing neurons to glucose.

(A) LK expression in the brain, illustrated by mCD8::GFP expression driven by LKGAL4. Scale bar represents 100 μm. (B) Representative traces and quantification of ex vivo calcium responses of LK+ neurons during the perfusion of glucose (n=6). (C-D) The distribution (C) and calcium responses (D) of TK+ neurons (n=6). (E-F) The distribution (E) and calcium responses (F) of CCHa1+ neurons (n=6). (G-H) The distribution (G) and calcium responses (H) of CCHa2+ neurons (n=6).

The calcium response of hugin neurons upon glucose stimuli.

(A) Representative image of hugin neurons in SEZ. Scale bar represents 50 μm. (B) Calcium responses of different cluster of hugin neurons during the prefusion of glucose (n=6)..*P < 0.05; ***P < 0.001. Student’s t-test used for comparisons.

hugin+ or AstA+ neurons suppressed sweet taste.

(A-D) Fraction of flies of the indicated genotypes and environmental temperatures showing PER to different concentrations of sucrose (n=4-5 groups, each of 10 flies). ns, P > 0.05; *P < 0.05; ***P < 0.001; ****P < 0.0001. Two-way ANOVA followed by post hoc test with Bonferroni correction was used for multiple comparisons when applicable.

The calcium response of AstA+ neurons upon glucose treatment.

(A) Representative image of different part of AstA neurons .Scale bar represents 50 μm. (B) Calcium responses of different cluster of hugin neurons during the prefusion of glucose (n=6)

Knockout of PK2-R1 enhanced sweet sensation.

(A-C) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). ***P < 0.001; ****P < 0.0001. Two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

hugin+ neurons in the brain activated AstA+ neurons.

(A-B) Fraction of flies of the indicated genotypes and environmental temperatures showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). Note connection between the brain and VNC was cut off before the behavioral assay. (C) Representative traces (upper) and quantification (lower) of peak calcium transients of AstA+ neurons after the photo-activation of hugin+ neurons (n=6) from ex vivo calcium imaging. Horizontal black bar represents the duration of red-light stimulation.

Manipulating gene AstA-R1 enhance PER.

(A-C) Fraction of flies of the indicated genotypes showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). ***P < 0.001; ****P < 0.0001. Two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

hugin-AstA-Gr5a circuitry inhibited feeding behavior.

(A) The illustration of experimental design in Figure supplementary 13B-C. Note that all flies were maintained at 20°C before the feeding assay to prevent neuronal silencing by Shibirets1 during these procedures. (B-C) Food consumption of flies of the indicated genotypes and environmental temperatures (n=8 groups, each of 4 flies). (D) Food consumption of flies of the indicated genotypes (n=8 groups, each of 4 flies). (E) The TG content of indicated genotypes (n=8 groups, each of 2 flies). (F) Food consumption of flies of the indicated genotypes (n=8 groups, each of 4 flies). ns, P > 0.05; ***P < 0.001; ****P < 0.0001. Student’s t-test and two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

NMU peptide suppressed sweet taste in fly.

Fraction of flies showing PER to different concentrations of sucrose (n=4 groups, each of 10 flies). Flies were injected with saline or synthetic NMU for 30 mins before the assay.**P < 0.01; ****P < 0.0001. Two-way ANOVA followed by post hoc test with Bonferroni correction were used for multiple comparisons when applicable.

The expressing pattern of NMU neurons and Calb2 neurons

(A) Co-labeling of NMU+ neurons using a Cre-dependent mCherry reporter (red) and in situ hybridization (green).Scale bar represents 200 μm. (B) NMU arbors was extended near Calb2rNST neurons. Scale bar represents 200 μm. (C) Co-labeling of Calb2+ neurons with a Cre-dependent mCherry reporter (red) and NMU antiboy staining (green). Scale bar represents 200 μm.

A working model

In the fly brain, a small group of neurons expressing hugin peptide is responsible for detecting the circulating glucose levels. Following feeding, as the levels of glucose in the circulatory system increased, these neurons are activated, leading to the release of hugin, which activated AstA neurons via its receptor PK2-R1. Subsequently, the activation of AstA+ neurons then directly inhibited sweet perception via AstA peptide and its cognate receptor AstA-R1 expressed in sweet-sensing Gr5a+ neurons. This neural circuitry is a novel energy sensor that suppressed sweet perception and terminated feeding behavior, providing an efficient mechanism for maintaining energy homeostasis.