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The gut–brain axis mediates sugar preference

Abstract

The taste of sugar is one of the most basic sensory percepts for humans and other animals. Animals can develop a strong preference for sugar even if they lack sweet taste receptors, indicating a mechanism independent of taste1,2,3. Here we examined the neural basis for sugar preference and demonstrate that a population of neurons in the vagal ganglia and brainstem are activated via the gut–brain axis to create preference for sugar. These neurons are stimulated in response to sugar but not artificial sweeteners, and are activated by direct delivery of sugar to the gut. Using functional imaging we monitored activity of the gut–brain axis, and identified the vagal neurons activated by intestinal delivery of glucose. Next, we engineered mice in which synaptic activity in this gut-to-brain circuit was genetically silenced, and prevented the development of behavioural preference for sugar. Moreover, we show that co-opting this circuit by chemogenetic activation can create preferences to otherwise less-preferred stimuli. Together, these findings reveal a gut-to-brain post-ingestive sugar-sensing pathway critical for the development of sugar preference. In addition, they explain the neural basis for differences in the behavioural effects of sweeteners versus sugar, and uncover an essential circuit underlying the highly appetitive effects of sugar.

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Fig. 1: Sugar activates the gut–brain axis.
Fig. 2: Silencing the sugar-activated circuit abolishes sugar preference.
Fig. 3: Vagal ganglion neurons transmit sugar signals to the brain.
Fig. 4: Imaging the gut–brain axis.
Fig. 5: Activation of sugar-responsive cNST neurons confers novel flavour preference.

Data availability

All data supporting the findings of this study are available from the corresponding author upon request.

Code availability

Custom code is available from the corresponding author.

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Acknowledgements

We thank N. Ryba for experimental suggestions and helpful comments; R. Barretto for advice on the calcium-imaging pipeline; L. Luo for the TRAP mice; S. Liberles for GPR65-Cre mice; P. Wulff for the tetanus toxin construct; A. Skowronski and C. Leduc for their assistance in performing blood glucose and insulin measurements; L. Rickman for expert help with figures; R. Lessard for earlier contributions; members of the Zuker lab for helpful discussions; and E. Sobolik, L. Hsin, Y. Zhang, A. Holguin, A. Conomikes, E. Shaw, B. McTyre and J. Li, who participated in various aspects of this work. Imaging was performed with support from the Zuckerman Institute’s Cellular Imaging platform. Research reported in this publication was supported in part by the Russell Berrie Foundation program in the neurobiology of obesity (to C.S.Z. and R. Leibel). A.C.S. was supported by the MSTP program, H.-E.T. was supported by the Agency for Science, Technology and Research (A*STAR) of Singapore, and Y.G. was supported by a predoctoral fellowship from NRSA and the MSTP program. C.S.Z. is an investigator of the Howard Hughes Medical Institute and a Senior Fellow at Janelia Farm Research Campus. Figures were generated with the help of BioRender.

Author information

Authors and Affiliations

Authors

Contributions

A.C.S. and H.-E.T. designed the study, carried out the experiments and analysed data, H.J. performed retrograde tracing experiments and helped with the TRAP system. M. Vignovich analysed calcium-imaging data, and helped to develop the analysis pipeline. M. Villavicencio and K.S.T. designed and characterized engineered animals and behavioural experiments. Y.G. participated in the initial phases of this study. C.S.Z. designed the study and analysed data. C.S.Z., A.C.S. and H.-E.T. wrote the paper.

Corresponding author

Correspondence to Charles S. Zuker.

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Competing interests

C.S.Z. is a scientific co-founder of and advisor to Kallyope. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Glucose and MDG preference.

a, When mice are given a choice between 600 mM glucose or 600 mM MDG, using a brief-access (1 h) test, naive animals display a small preference for glucose over MDG (n = 5, two-tailed paired t-test, P = 0.0406), probably because MDG is slightly less sweet and thus not as attractive. Values are mean ± s.e.m. b, c, Although the non-caloric sugar analogue MDG is very effective in causing a preference switch (see Fig. 1), it does not cause increases in plasma glucose or release of insulin. Mice were gavaged with glucose or MDG, and plasma glucose and insulin levels were sampled before (Pre), and at 15 min after the gavage (Post). b, Plasma glucose after glucose gavage (red bars). n = 7, two-tailed paired t-test, P = 4 × 10−5. Plasma glucose after MDG gavage (blue bars). n = 6, two-tailed paired t-test, P = 0.36. c, Plasma insulin levels after glucose gavage (red bars). n = 7, two-tailed paired t-test, P = 7 × 10−6. Plasma insulin levels after MDG gavage (blue). n = 6, two-tailed paired t-test, P = 0.94. Values are mean ± s.e.m.

Extended Data Fig. 2 Fos responses are robust and reliable.

a, The brain diagram illustrates the position of the NST and the plane of the sectioning. Shown are cNST sections stained with Fos antibodies after exposing the animals to 90 min of 600 mM sucrose, 600 mM glucose or 30 mM AceK. Each panel is a confocal maximal projection image from Bregma −7.5 mm consisting of 3 sections 15 μm apart. Each panel (sucrose, glucose or AceK) represents a different animal, n = 3 independent experiments. Note the robustness of the signals across animals. See Methods for details. b, Mice were stimulated with 600 mM 3-OMG (n = 6 mice) or 600 mM galactose (n = 3 mice) (see also Fig. 4, Extended Data Fig. 10). Note strong Fos signals in cNST neurons, n = 2 independent experiments (total of 9 mice). Scale bars, 100 μm.

Extended Data Fig. 3 The development of sugar preference.

a, Glucose stimulates cNST neurons in mice lacking the sweet taste receptor (T1R2/3−/−), or in mice lacking the TRPM5 ion channel (TRPM5−/−). See Fig. 1e for quantification. T1R2/3−/−, n = 5 mice, ANOVA followed by Tukey’s HSD post hoc test, P < 0.0001; TRPM5−/−, n = 7 mice, ANOVA followed by Tukey’s HSD post hoc test, P < 0.0001. Values are mean ± s.e.m. Scale bars, 100 μm. b, Direct intragastric infusion of glucose, but not AceK, robustly activates the cNST. n = 2 independent experiments. Scale bars, 100 μm. c, d, Genetic silencing of vagal sensory neurons. c, Sugar-preference graphs for wild-type mice (n = 5 mice), demonstrating the robust development of preference for sugar versus artificial sweetener (see also Fig. 1). By contrast, silencing of the sensory neurons in the nodose ganglia, by bilateral injection of AAV-DIO-TetTox into the nodose ganglia of Vglut2-cre mice (see Methods), abolishes the development of sugar preference; n = 3 mice, two-sided Mann–Whitney U-test, P = 0.035. Values are mean ± s.e.m. d, However, silencing vagal sensory neurons does not impair the innate attraction to sweet solutions; shown are behavioural responses to AceK versus water, and glucose versus water (n = 3 animals, preference index for AceK = 0.82, preference index for glucose = 0.85). Values are mean ± s.e.m.

Extended Data Fig. 4 Retrograde labelling from cNST.

a, A fluorescent retrograde tracer (red RetroBeads, Lumafluor) was stereotactically injected into the cNST to label its inputs. The nodose ganglia and dorsal root ganglia were checked for transfer of the fluorescent label after 6–7 days. The nodose ganglion (vagal neurons), but not the dorsal root ganglion (spinal neurons), was robustly labelled60. n = 2 independent experiments. b, RetroBeads were also injected into the cuneate nucleus, a brainstem area near but distinct from the cNST. Vagal neurons were not labelled. By contrast, note robust labelling of spinal neurons (n = 2 independent experiments). Nuclei were counterstained with DAPI (blue). Scale bars, 200 μm (Brainstem), 50 μm (nodose, DRG). c, Validation of TRAPing procedure to confirm that the sugar-activated cNST neurons marked by the expression of Fos are the same as the ones labelled by Cre recombinase in the genetic TRAPing experiments. We genetically labelled the sugar-induced TRAPed neurons with a Cre-dependent fluorescent reporter61, and then performed a second cycle of sugar stimulation followed by Fos antibody labelling. d, Top, neurons labelled by the Cre-dependent reporter after sugar TRAPing (‘sugar-TRAP’, pseudocoloured red) are the same as those labelled by Fos after a second cycle of sugar stimulation (‘sugar-Fos’, green; see Methods and text for details), >80% of Sugar-Fos neurons are also sugar-TRAP positive (n = 7 animals). Middle, note that the few neurons labelled after water-TRAP in response to water do not overlap with those labelled with Fos antibodies after sugar stimulation. Bottom, the sugar-TRAP neurons are also activated by the non-caloric sugar analogue MDG; >80% of MDG-Fos are sugar-TRAP positive. Scale bar, 20 μm.

Extended Data Fig. 5 Mice with a silenced sugar-preference circuit behave as normal mice, drinking artificial sweeteners.

a, A normal, non-thirsty mouse drinks about 5 ml of water during a 24-h window. n = 11 mice. Values are mean ± s.e.m. b, If presented with a sweet option (but not sugar, so as to not create a preference) they show a small but significant increase in total volume consumed, but now most of the total consumption is from the sweet choice rather than water (n = 9 animals, two-tailed paired t-test, P = 1 × 10−4). Values are mean ± s.e.m. c, By contrast, if the options are water versus sugar, so that it creates a preference, they massively increase total volume consumed, and nearly all is from the sugar solution (n = 9 animals, two-tailed paired t-test, P = 3 × 10−10). Values are mean ± s.e.m. d, As expected, wild-type controls develop a strong preference for sugar versus AceK (n = 9 animals, two-tailed paired t-test, P = 3 × 10−8). Values are mean ± s.e.m. e, f, Mice with the preference circuit silenced behave as control animals presented with a sweet, non-preference creating choice (compare ef with b) (n = 6 mice, two-tailed paired t-test, P = 6 × 10−4 for AceK, P = 4 × 10−3 for glucose). Values are mean ± s.e.m. g, Silenced animals consumed nearly equal volumes of sugar and artificial sweetener (n = 6 animals, two-tailed paired t-test, P = 0.1). Values are mean ± s.e.m.

Extended Data Fig. 6 Vagal-neuron responses to sugar and MDG are highly reproducible and timed-locked to the stimulus.

a, Shown are vagal-neuron responses to 6 consecutive 10-s intestinal stimuli of alternating trials with 500 mM glucose and 500 mM MDG (stimulus delivery and timings are as described in the Methods). Each of the sample traces depicts the response from a different neuron. b, Shown are vagal-neuron responses to 5 consecutive 10-s intestinal stimuli with 500 mM glucose (stimulus delivery and timings are as described in the Methods). Each of the sample traces shows the response from a different neuron. c, Expanded time scale of responses to the 10-s 500 mM glucose stimulus from 10 s before to 10 s after termination of the stimulus. The green dashed lines indicate the initiation of the stimulus, and the blue dashed lines denote termination of the 10-s stimulus. Calcium responses are shown in solid black and exponential fits to the response latency and kinetics are shown in red. Note responses time-locked to stimulus delivery; the top two traces depict two cells from two different mice in response to glucose, and the bottom two traces depict two cells from two different mice in response to MDG; latencies varied between 3 and 6 s, and were within the 10-s stimulation window. Some cells exhibited longer latencies (see for example, heat maps in Fig. 4, Extended Data Fig. 8). We believe the cells with longer response latencies may represent intestinal glucose responders located farther down the intestinal segment, and thus would be expected to demonstrate longer latencies37. d, On average, approximately 5% of vagal neurons respond reliably to a 10-s 500 mM glucose stimulus. The histogram shows the percentage of GCaMP-expressing vagal neurons responding to the 10-s glucose stimulus. Average = 4.6 ± 0.05% (n = 4,803 neurons from 51 ganglia, mean ± s.e.m.). e, Recent findings25 have suggested that appetitive behavioural responses are elicited through stimulation of vagal terminals originating from the right nodose ganglion. Shown are heat maps depicting z-score normalized average calcium responses of individual ganglion neurons after a 60-s pulse of 500 mM glucose. We observe no differences in responses to intestinal glucose from either the left or right vagal ganglia. Also shown are example traces from different neurons from the left and right Nodose ganglion; red bars indicate the 60-s stimulus; scale bars indicate percentage maximal response.

Extended Data Fig. 7 Vagal neurons innervating duodenal segment sense sugar.

a, Top, schematic of retrograde tracing experiment. Fluorescently conjugated CTB38 was injected into the proximal duodenum to back fill and label the cell bodies of duodenum-projecting vagal neurons (z-projection of n = 22 confocal planes from a representative ganglion, see Methods for details). The two bottom panels show a sample retrogradely labelled ganglion with sensory neurons (Vglut2-cre driving the GCaMP reporter) marked in green (left) and those labelled by CTB marked by red fluorescence (right). Double-positive neurons are highlighted by the white circles. Scale bar, 100 μm. b, Representative field of a vagal imaging session showing the overlay of CTB and GCaMP. The two yellow circled neurons (denoted as #1 and #2) were labelled by retrogradely applied CTB in the duodenal segment, and exhibited strong responses to glucose (n = 16 ganglia from 10 mice). Scale bar, 100 μm. c, A total of 12 out of 55 double-positive neurons responded to the 10-s glucose stimulus (see Extended Data Fig. 6d for a comparison with uninjected animals). n = 16 ganglia from 10 mice. Note the substantial enrichment in the number of responders when pre-tagged by retrograde labelling: ~20% in the duodenal tagged versus 4–5% in the whole population.

Extended Data Fig. 8 Glucose responders are not sensing osmolarity.

Williams et al.24 identified vagal neurons that indiscriminately responded to high concentrations of several stimuli delivered in very large stimulus volume for hundreds of seconds. We believe these responses, largely independent of the quality of the stimulus, are intestinal osmolarity signals. a, Shown are heat maps summarizing responses to interleaved 60-s stimuli of 500 mM glucose and 500 mM mannitol. Each row represents the average activity of a single cell during three interspersed exposures to the stimulus. Stimulus window is indicated by the dashed white lines. Of 134 neurons that responded to intestinal application of 500 mM glucose for 60 s, 101 did not exhibit statistically significant responses to mannitol (top). However, 33 (~25%) showed responses to both 500 mM glucose and 500 mM mannitol (bottom). n = 5 mice. When the intestinal stimulus consisted of a short pulse (that is, 10 s; 33 μl volume) no responses were detected to 500 mM mannitol (data not shown). b, Sample traces (three trials each) of a neuron responding to glucose (red) but not mannitol (blue). c, Sample traces (three trials each) of a neuron responding to glucose and mannitol. Scale bars indicate percentage maximal response. d, Heat maps showing responses to a 60-s stimuli of 1 M mannitol, 1 M fructose, 1 M mannose and 1 M NaCl. Note that the same cells respond indiscriminately to the various stimulus (n = 4 mice). e, The graph shows preference plots for fructose versus AceK (n = 8 mice, two-tailed paired t-test, P = 0.27). Note that fructose, a caloric sugar, does not create preference, but activates osmolarity responses. f, Williams et al.24 suggest that GPR65-expressing vagal neurons function as the nutrient sensors. We generated mice in which GCaMP6s expression was targeted to GPR65-expressing vagal neurons and examined their responses to a 10-s stimulus of 500 mM glucose or osmolarity signals (that is, 1 M each of fructose, mannose and NaCl for 60 s). Shown are normalized responses of from three different mice to the four stimuli; each trace represents a different responding neuron. Note that 500 mM glucose for 10 s does not activate GPR65 neurons. By contrast, they are activated by 60-s pulse of 1 M fructose, mannose and NaCl (see also Fig. 4). g, Summary histogram of GPR65 tuning profile to 10 s 500 mM glucose, and 60 s 1 M fructose, 60 s 1 M mannose and 60 s 1 M NaCl; n = 4 mice.

Extended Data Fig. 9 Genetic silencing of GPR65 neurons does not affect the development of sugar preference.

a, Global silencing of the GPR65 neurons was achieved by generating GPR65-IRES-Cre; R26-TeNT double transgenic animals expressing TetTox in GPR65 neurons. Sugar-preference graphs demonstrating the robust development of preference for sugar versus artificial sweetener for both wild-type (n = 5 mice, two-tailed paired t-test, P = 0.0047) and GPR65:TetTox mice (n = 5 mice, two-tailed paired t-test, P = 0.0033). The wild-type controls shown here are the same mice used in Extended Data Fig. 3c, as both sets of silencing experiments were carried out as part of the same series of studies. Values are mean ± s.e.m. b, Silencing of GPR65 neurons does not impair the innate attraction to sweet solutions. Shown are behavioural responses to AceK versus water and glucose versus water (n = 5, two-tailed paired t-test, P = 0.0040 for consumed volumes of AceK versus water, P = 0.0023 for consumed volumes of glucose versus water). Values are mean ± s.e.m.

Extended Data Fig. 10 Vagal neurons responding to intestinal glucose are also activated by SGLT1 agonists.

a, Traces of vagal neurons responding to a 10-s pulse of 500 mM intestinal glucose, also challenged with a 10-s pulse of 500 mM 3-OMG. Shown are sample neurons from 2 animals. b, Traces of vagal neurons responding to a 10-s pulse of 500 mM intestinal glucose, also challenged with a 10-s pulse of 500 mM galactose. Shown are sample neurons from two animals for expanded time scales (from Fig. 4d). c, Traces of vagal neurons responding to a 10-s pulse of 500 mM intestinal glucose, also challenged with a 10-s pulse of 500 mM fructose and 500 mM mannose. Shown are sample neurons from three mice. d, Traces of vagal neurons responding to two consecutive 10-s pulses of 500 mM intestinal glucose, before and after treating the intestinal segment with 8 mM phlorizin for 5 min. Note the loss of responses. e, Because responses, in general, show some decay during the time of the experiment (in part due to desensitizing and bleaching of the fluorescent signals), we also analysed the average decay of corresponding glucose responses in the absence of any blocker. The graphs compare the loss of responses during normal decay, and in response to the blocker. For normal decay (left), n = 11 neurons, Pre = 230.8 arbitrary units (a.u.), Post = 172.8 a.u.; for blocker (right), n = 31 neurons, Pre = 229.7 a.u., Post = 67.0 a.u. All values are mean ± s.e.m. Scale indicates average integral of the responses to the two trials before and after inhibition.

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Tan, HE., Sisti, A.C., Jin, H. et al. The gut–brain axis mediates sugar preference. Nature 580, 511–516 (2020). https://doi.org/10.1038/s41586-020-2199-7

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