Symmetric brain-liver circuits mediate lateralized regulation of hepatic glucose output

Zhonglong Wang; Xiangfei Gong; Li Jiang; Ke Wang; Xinyuan Sun; Yingxi Li; Mengtong Ran; Yanshen Chen; Hongdong Wang; Xuehui Chu; Shun Wang; Junjie Wang; Xiao Zheng; Haiping Hao; Hao Xie

doi:10.7554/eLife.109599.1

eLife Assessment

This manuscript proposes a lateralized, lobe-specific brain-liver sympathetic neurocircuit regulating hepatic glucose metabolism and presents anatomical evidence for sympathetic crossover at the porta hepatis using viral tracing and neuromodulation approaches. While the topic is of important significance and the methodologies are, in principle, state-of-the-art, significant concerns regarding experimental design, incomplete methodological reporting, sparse and ambiguous labeling, and overi-nterpretation of the data substantially weaken support for the study's central conclusions, thereby limiting the study's completeness. The work will be of interest to biologists, clinicians, and physiologists.

https://doi.org/10.7554/eLife.109599.1.sa5

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Abstract

Hemispheric lateralization is well recognized in regulating contralateral somatic movement, yet its relevance to visceral organ regulation remains poorly understood. This study aims to investigate whether cerebral hemispheres differentially regulate hepatic glucose metabolism and localize the site of sympathetic crossover to the liver. Pseudorabies virus (PRV) tracing demonstrated symmetric projections from the paragigantocellular nucleus (LPGi) with preferential innervation of contralateral hepatic lobes. Unilateral LPGi activation elevated systemic glucose through enhanced glycogenolysis and gluconeogenesis in contralateral lobes, whereas bilateral activation produced additive effects. Following unilateral hepatic denervation, contralateral LPGi activation induced metabolic compensation in the remaining innervated lobes, characterized by increased sympathetic release, glucose production, and glycogen depletion. Whole-mount clearing and dual tracing localized the sympathetic crossover to the porta hepatis, and developmental analysis showed lobar innervation along the vasculature emerging at postnatal week 2. These findings demonstrate that the brain exerts lobe-specific, lateralized control of hepatic glucose metabolism via symmetric brain-liver sympathetic pathways. Contralateral regulation arises from peripheral decussation at the porta hepatis, and compensatory activation following denervation reveals an intrinsic neuroadaptive mechanism that safeguards systemic glucose homeostasis.

Highlights

Brain-liver sympathetic projections exhibit predominant contralateral innervation
Unilateral LPGi activation drives glucose production in contralateral hepatic lobes
Unilateral denervation augments contralateral LPGi-mediated metabolic compensation
Sympathetic crossover to the liver localizes at the porta hepatis

Introduction

The cerebral hemispheres exhibit pronounced functional asymmetry,^(1,2) a phenomenon traditionally associated with cognitive and motor processes such as language, voluntary movement, and spatial navigation.^(3,4) Emerging evidence indicates that this lateralization extends beyond cortical functions to the autonomic regulation of visceral organs, thereby enhancing the efficiency of physiological responses.^(5–7) Such hemispheric specialization allows the central nervous system (CNS) to optimize resource allocation and prioritize functional outputs in a context-dependent manner. For instance, paired organs such as kidneys and adrenal glands receive predominantly contralateral neural inputs, enabling coordinated bilateral function.^(8,9) Previous studies have shown that the left hemisphere preferentially modulates sympathetic outflow to the right kidney and vice versa,^(8,10) and that cortical projections to the adrenal medulla are primarily contralateral, with the rostral cingulate motor area as a bilateral exception.⁽⁹⁾ However, the influence of hemispheric asymmetry on structurally asymmetric visceral organs remains incompletely understood and appears to be organ-specific. The heart, for instance, exhibits distinct lateralized autonomic innervation: right-sided fibers predominantly mediate chronotropic regulation via the sinoatrial node, whereas left-sided fibers exert greater inotropic and dromotropic functions.^(6,11) Additionally, cortical neurons regulating gastric sympathetic function are predominantly localized in the right hemisphere, whereas parasympathetic control displays only a modest right-sided predominance.^(7,12) These examples underscore the organ-specific nature of hemispheric asymmetry in autonomic neural projections.

The liver, as the largest asymmetrical visceral organ, plays a central role in systemic metabolic homeostasis,^(13,14) orchestrating physiological processes such as glucose production, lipid metabolism, protein synthesis, and detoxification.^(16,17) In mice, hepatic innervation first appears in extrahepatic bile ducts at embryonic day 17.5 and extends into lobes during the early postnatal period,⁽¹⁸⁾ yet the precise pattern of this innervation remains unknown. Previous anatomical and functional studies, including our own, have established that the liver receives dense sympathetic innervation from postganglionic neurons of the celiac-superior mesenteric ganglia (CG-SMG).^{(19,20,21,22)} Within this context, the brainstem lateral paragigantocellular nucleus (LPGi) has been identified as a key node in the regulation of hepatic sympathetic tone and glucose output. However, whether this regulation is lateralized and lobe-specific, and where sympathetic crossover occurs, has not been systematically defined.

To address these gaps, we applied retrograde trans-synaptic tracing with pseudorabies virus (PRV) to delineate LPGi projections to hepatic lobes and assess their lateralization.⁽²²⁾ Functional experiments employing chemogenetic and optogenetic activation of LPGi neurons clarified the physiological impact of this neural circuitry on hepatic glucose production (HGP). Additionally, we identified compensatory neural mechanisms that maintain systemic glucose homeostasis following unilateral cerebral disruption of hepatic innervation. Whole-mount clearing identified the porta hepatis as the site of sympathetic crossover and revealed developmental axon extension of hepatic fibers along the vasculature. Together, these findings reveal that lateralized neuronal circuits exert lobe-specific control over hepatic glucose output and recruit neuroadaptive responses to preserve metabolic homeostasis following unilateral disruption.

Results

1. Contralateral projections from the LPGi to distinct hepatic lobes

Previous studies of intrahepatic innervation mainly focused on individual lobes, such as the left or right lateral lobes in mice.^(14,15) We first asked whether sympathetic innervation is a uniform feature across all lobes. To address this, we examined the intact mouse liver using tissue clearing combined with immunofluorescence staining of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. This analysis revealed that, although hepatic lobes differ in volume (Figure 1A), sympathetic fibers consistently branch into all lobes with comparable density (Figure 1A). To further investigate the anatomical basis of central neural regulation of the liver, we performed multi-synaptic retrograde tracing by injecting PRV into individual hepatic lobes, including the left lateral, median, right posterior, right anterior, and caudate lobes (Figures S1A and S1B). Five days post-injection, PRV-labeled neurons were consistently identified in the LPGi of the brainstem (Figure 1B, Figures S1C-S1F). Quantitative analyses revealed a clear contralateral dominance: the injection into left lateral and median lobes yielded approximately twice as many labeled neurons in the right LPGi as in the left, whereas the injection into right posterior, right anterior, and caudate lobes preferentially labeled neurons in the left LPGi (Figure 1C). These findings provide direct anatomical evidence of a symmetric projection pattern in which each LPGi hemisphere preferentially innervates contralateral hepatic lobes (Figure 1D). Based on their robust lateralization, the right anterior and median lobes were selected as representative targets for subsequent analyses.

The brain contralaterally projects to the liver.
(A) Representative 3D images of TH-immunostained mouse liver (left), with quantification of sympathetic axon density and lobe volume (right). Scale bar, 3,000 μm. (B) Representative immunofluorescence images of PRV-labeled neurons (EGFP) in the left (top) and right (bottom) LPGi following PRV injections into the right anterior (left) and median (right) lobes. Scale bars, 100 μm. (C) Quantification of PRV-labeled neurons in left and right LPGi across different hepatic lobes: left lateral, median, right posterior, right anterior, caudate, and porta hepatis (n = 3). (D) Sankey diagram showing projection patterns from left and right LPGi to individual hepatic lobes. (E and F) Representative slices of EGFP⁺ and mRFP⁺ neurons in left (top) and right (bottom) LPGi following PRV-EGFP (right anterior lobe) and PRV-mRFP (median lobe) injections. Proportions of EGFP⁺, mRFP⁺, and co-labeled neurons in left and right LPG (F, n = 3). Scale bars, 100 μm. Data are expressed as means ± SEM, with individual values shown for all experiments. *p < 0.05, **p < 0.01. Unpaired Student’s t-test for (C).

To validate these projection patterns, we performed dual labeling by co-injecting PRV-CAG-EGFP into the right anterior lobe and PRV-CAG-mRFP into the median lobe. In the left LPGi, 73.7% of PRV-positive neurons expressed EGFP, showing projections to the right anterior lobe, whereas in the right LPGi, 62.5% expressed mRFP, indicating projections to the median lobe (Figures 1E and 1F). A subset of neurons displayed co-labeling, suggesting that although the predominant projection pattern is contralateral, a population of LPGi neurons innervates both lobes bilaterally. These findings establish a contralaterally biased projection pattern from each LPGi to distinct hepatic lobes, providing the anatomical foundation for lateralized brain-liver circuits that regulate hepatic glucose metabolism.

2. Differential HGP by LPGi lateralization

To assess the functional implications of this anatomical laterality, we examined whether activation of the LPGi in each hemisphere modulates HGP in a lobe-specific manner. Building on our previous findings that liver-associated LPGi neurons are predominantly GABAergic, we selectively activated these neurons unilaterally or bilaterally using chemogenetics (Figure 2A, Figure S2A). LPGi activation significantly increased blood glucose, with area under the curve (AUC) increases by 27%, 34%, and 52% following left-, right-, and bilateral stimulation, respectively (Figure 2A). Moreover, right-sided activation induced greater hyperglycemia than left-sided stimulation, possibly reflecting differences in the metabolic capacity or volume of the contralateral hepatic lobes (Figure 1A).

LPGi stimulation raises blood glucose by driving contralateral, lobe-specific hepatic glycogenolysis.
(A) Scheme for injecting AAV-DIO-hM3Dq or AAV-DIO-mCherry (top). Blood glucose levels following chemogenetic activation of the left, right, or bilateral LPGi, measured at −10, 30, 60, 90, and 120 min (bottom, n = 6). (B and C) Representative immunofluorescence images of pGS (B) and pPYGL (C) expression in the median (top) and right anterior (bottom) lobes after 1 h chemogenetic activation of left, right, or bilateral LPGi, with quantification of relative fluorescence intensity (n = 5). Scale bars, 100 μm. (D) Western blot analysis of pPYGL, PYGL, pGS, and GS in median (left) and right anterior (right) lobes after 1 h activation, with densitometric quantification (bottom, n = 3). (E) Plasma concentrations of norepinephrine (NE) (left, n = 5) and epinephrine (EPI) (right, n = 5) following 1 h activation. (F) NE contents in median (left, n = 5) and right anterior (right, n = 5) lobes after 1 h activation. Data are expressed as means ± SEM, with individual values shown for all experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; ^$$$p < 0.001. One-way ANOVA with the Tukey test for (A -F).

At the molecular level, one hour following stimulation, phosphorylation of glycogen phosphorylase (pPYGL) and glycogen synthase (pGS) increased in contralateral lobes, as assessed by immunofluorescence and western blot. Left LPGi activation preferentially enhanced pPYGL and pGS levels in the right anterior lobe compared with right-sided activation, whereas right LPGi activation exerted a greater effect in the median lobe relative to left-sided stimulation (Figures 2B-2D). Bilateral stimulation further amplified pPYGL and pGS expression in both lobes compared with unilateral activation. PAS staining confirmed glycogen depletion in metabolically engaged contralateral lobes (Figure S2B). In terms of gluconeogenesis, enzymatic activity assays revealed increased phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6PC) activity in hepatic lobes undergoing enhanced glycogenolysis, despite unchanged protein levels within the one-hour window (Figures S2C-S2F).

To exclude systemic endocrine contributions, particularly via the hypothalamic-pituitary-adrenal (HPA) axis, we measured plasma NE and EPI 1 hour after LPGi activation. Neither hormone changed significantly following unilateral nor bilateral stimulation (Figure 2E). In contrast, intrahepatic NE content rose specifically in contralateral lobes: by 1.49-fold in the right anterior lobe after left LPGi activation, 1.53-fold in the median lobe after right LPGi activation, and over 1.85-fold in both lobes after bilateral activation (Figure 2F). These findings support a mechanism whereby lobe-specific sympathetic activation drives hepatic glucose output in a lateralized manner.

To achieve higher temporal precision, we applied optogenetics by delivering AAV9-GAD67-Cre with AAV9-DIO-ChR2-mCherry viruses to the LPGi (Figure S3A, Figure S4A). Compared with chemogenetics, optogenetic activation elicited greater hyperglycemia, with AUC rising by 32%, 42%, and 62% for left-, right-, and bilateral stimulation, respectively (Figure S3A). Molecular and histological analyses paralleled those from chemogenetics, including contralateral and additive effects on pPYGL and pGS (Figures S3B-S3D), glycogen depletion (Figure S4B), elevated intrahepatic NE content (Figure S3F), and enhanced PEPCK and G6PC activity (Figures S4C-S4F), without changes in plasma catecholamines (Figure S3E). Taken together, these findings demonstrate that each LPGi hemisphere preferentially drives glucose production in contralateral hepatic lobes, primarily through localized sympathetic activation and glycogenolysis in contralateral hepatic regions.

3. Compensatory neural responses following unilateral hepatic denervation

While compensatory adaptations following unilateral damage are well described in symmetrical organs, it remains unclear whether similar mechanisms operate in asymmetrical organs such as the liver.^(26–28) To test this, we chemically denervated the right- or left-sided lobes using 6-OHDA. Despite the absence of directly sympathetic input to denervated lobes, systemic blood glucose levels were unchanged compared with controls (Figures 3A-3C, Figure S5A), indicating functional compensation through the remaining intact liver. Further analyses revealed increased expression of pPYGL and pGS, as well as elevated intrahepatic NE levels and glycogen depletion in non-denervated hepatic lobes (Figures 3D-3H, Figures S5B-S5E).

LPGi maintains glucose balance through compensatory use of ipsilateral hepatic lobes under contralateral lobe impairment.
(A and B) Blood glucose levels in the left- (A, n = 7) or right-sided (B, n = 7) lobes denervated mice. Western blot of TH protein in median and right anterior lobes after denervating left- (top) or right-sided (bottom) lobes. (D and E) Representative immunofluorescence images of pGS (top) and pPYGL (bottom) expression in right anterior and median lobes in left- (D) or right-sided (E) lobes denervated mice, with quantification of relative fluorescence intensity (right, n = 5). Scale bars, 100 μm. (F and G) Western blot analysis of pPYGL, PYGL, pGS, and GS proteins in median and right anterior lobes (top) in left- or right-sided lobes denervated mice, with densitometric quantification (bottom, n = 3). (H) NE contents of median and right anterior lobes in left- or right-sided lobes denervated mice (n = 6). (I) Representative immunofluorescence images of c-FOS expression in left and right LPGi in left- or right-sided lobes denervated mice. Scale bars, 100 μm. Data are presented as means ± SEM, with individual values shown for all experiments. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired Student’s t-test for (A-B, D-H).

To elucidate the underlying mechanism, we quantified c-FOS expression in the LPGi following unilateral hepatic denervation. Right-sided lobes denervation increased c-FOS in the left LPGi, and vice versa (Figure 3I), suggesting that contralateral LPGi upregulates its sympathetic output to intact ipsilateral hepatic lobes, thereby compensating for the loss of innervation in denervated lobes. Together, these findings identify a neuroadaptive mechanism in which the ipsilateral LPGi augments sympathetic output to intact hepatic lobes, thereby maintaining systemic glucose homeostasis despite partial intrahepatic neural disruption.

4. Peripheral decussation of sympathetic hepatic control at the porta hepatis

Contralateral neural circuitry typically arises from central decussations, such as corticospinal tracts that cross at the pyramids for limb motor control^(29,30) and visual pathways that decussate at the optic chiasm for hemispheric processing of visual fields.⁽³¹⁾ Whether the brain’s contralateral regulation of the liver occurs centrally or peripherally, however, has remained unknown. Using whole-mount clearing, we reconstructed the brain-liver sympathetic pathway and found that central projections directly descend from the brainstem along the sympathetic chain (SyC) to the CG-SMG, which then sends postganglionic fibers to the liver (Figure 4A). This anatomical arrangement suggested that contralateral control might arise from a peripheral decussation from the CG-SMG to the liver. To test this hypothesis, we transparentized the mouse trunk and observed that sympathetic nerve bundles from the CG-SMG tracked along the hepatic portal vein deep into the liver (Figure 4B). We speculate that these nerve bundles decussate in the hepatic portal region before extending into the individual liver lobes.

Sympathetic projections to the liver decussate at the porta hepatis, progressively formed during development.
(A) Representative 3D images of mice following PRV injection into right- (left) or left-sided (right) lobes. Scale bars, 3000 µm. (B) Representative 3D images of CG-SMG projections to peripheral organs. Scale bar, 3,000 μm. (C) Representative images of sympathetic axons after unilateral CG injection, with quantification of EGFP-labeled axon distribution across hepatic lobes. Scale bars, 100 μm. (D) Representative images of WGA-labeled neurons in left and right CG, with percentages of WGA555⁺, WGA647⁺, and co-labeled neurons. Scale bars, 100 μm. (E) Representative images showing the developmental progression of CG-SMG projections to the liver from postnatal week 0 to week 2. Scale bars, 3,000 µm.

To test this, we performed unilateral anterograde tracing by injecting AAV-TH-DIO-EGFP into a single CG (Figure S5F). This revealed a strictly contralateral innervation pattern: the left CG mainly projected to the right posterior, right anterior, and caudate lobes, whereas the right CG prominently innervated the left lateral and median lobes (Figure 4C). To validate this pattern, we performed dual retrograde tracing by injecting Alexa Fluor 647- and 555-conjugated wheat germ agglutinin (WGA) into left- and right-sided lobes, respectively (Figure S5G). Consistent with the anterograde results, left CG neurons predominantly projected to the right-sided lobes, while right CG neurons targeted the left-sided lobes, confirming robust contralateral preference (Figure 4D). Thus, these findings demonstrate that central inputs descend via the SyC to the CG-SMG complex, where nerve bundles converge and extend along the portal vein, branching within the porta hepatis to innervate individual hepatic lobes.

To explore the development of the intrahepatic neural network in mice, we therefore performed tissue clearing on the torsos of mice from postnatal weeks 0 to 2. We observed that at week 0, the sympathetic nerve bundles were located at the porta hepatis but had not yet extended into the liver (Figure 4E, left). By week 1, the sympathetic nerves had begun to grow into the liver (Figure 4E, middle). It was not until week 2 that the sympathetic nerves fully infiltrated all liver lobes (Figure 4E, right). These findings indicate that, after the first postnatal week, sympathetic nerves grow from the porta hepatis into the liver lobes and form a well-developed sympathetic neural network by the second week.

Discussion

The brain exhibits marked functional asymmetry despite structural bilateral symmetry, a phenomenon well recognized in language, handedness, and spatial cognition.^(32,33) Recent advances extend this concept beyond cortical domains to central autonomic regulation of peripheral organs.^(34,35) Trans-neuronal tracing of symmetrical organs such as the kidneys and thymus has revealed contralateral control with bilateral coordination,^(8,33) whereas the lateralized regulation of asymmetrical organs like the liver, heart, and stomach remains poorly defined and conflicting.^(6,12,37) For instance, parasympathetic cardiac control has been reported to exhibit left-hemispheric dominance,⁽⁶⁾ yet clinical evidence revealed that arrhythmias may arise after strokes in either hemisphere.⁽³⁸⁾ These discrepancies highlight the complexity of lateralized autonomic control and the intrinsic bilaterality within the autonomic network. In this study, we systematically mapped and functionally interrogated the laterality of brain-liver circuitry, focusing on the LPGi. Our anatomical and functional experiments provide compelling evidence that although a subset of LPGi neurons project bilaterally, the predominant organization is contralateral and lobe-specific.

Recent advances in viral tracing and whole-tissue imaging have greatly accelerated our understanding of peripheral neuroanatomy.^(39–41) For instance, distinct gastrointestinal regions are differentially regulated by subdivisions within the CG-SMG,⁽¹⁹⁾ while the kidneys predominantly receive ipsilateral input from sympathetic postganglionic neurons residing in the renal sympathetic ganglia.⁽²⁰⁾ In contrast, components of the immune system, such as the thymus, exhibit lateralized central regulation, with each hemisphere preferentially modulating the contralateral thymic lobe.⁽³⁶⁾ Using PRV-mediated retrograde tracing with whole-mount clearing, we visualized a brain-liver axis originating from the LPGi and revealed a strikingly lateralized projection pattern: left LPGi neurons preferentially innervated right-sided lobes, whereas right LPGi neurons predominantly targeted left-sided lobes. A smaller subset projected bilaterally, suggesting a degree of cooperative regulation across hemispheres. This high-resolution anatomical mapping provides direct evidence for lateralized central projections to an asymmetrical organ, underscoring a previously unrecognized dimension of laterality in autonomic liver innervation.

Functionally, this organization translates into lobe-specific regulation of HGP. Sympathetic innervation of the liver is well established as a key driver of glucose output, with prior electrical stimulation studies demonstrating robust hepatic glucose release in response to sympathetic activation.^(42–44) Consistent with these observations, both chemogenetic and optogenetic activation of LPGi GABAergic neurons significantly elevated systemic glucose levels by promoting glycogenolysis and gluconeogenesis in contralateral lobes. These metabolic effects were accompanied by localized NE release, increased phosphorylation of key metabolic enzymes (pPYGL and pGS), and glycogen depletion.^(45,46) Notably, bilateral LPGi activation produced additive effects, indicating that both hemispheres can cooperate to augment hepatic metabolism, but in a spatially segregated manner. However, whether such asymmetric input also extends to other hepatic functions, including lipid metabolism, protein synthesis, or detoxification, remains to be determined.⁽¹³⁾

Beyond identifying lateralized control, we also reported a robust compensatory mechanism following unilateral hepatic denervation. When neural input from one hemisphere was attenuated, compensatory neural plasticity emerged through increased sympathetic drive from contralateral LPGi, enhancing gluconeogenesis and glycogenolysis within the remaining innervated hepatic lobes. This response was supported by increased c-FOS expression in contralateral LPGi, higher intrahepatic NE content, and elevated pPYGL and pGS expression in unaffected lobes. Such plasticity parallels compensatory phenomena in other systems, including contralateral enhancement after renal dysfunction and cortical dominance shifts in patients with unilateral hearing loss.^(26,47) Our findings suggest that the brain-liver axis possesses a similar adaptive capacity, enabling preservation of systemic glucose homeostasis and potentially other hepatic functions (e.g., lipid metabolism, bile secretion) despite partial loss of hepatic innervation.

We further show that contralateral regulation of hepatic metabolism arises from a peripheral decussation, challenging the long-held view that such organization arises exclusively from central crossings. Unlike somatic and sensory systems, where contralateral control is mediated by pyramidal or optic decussations in CNS nuclei,^(2,26,27) hepatic sympathetic projections descend ipsilaterally from the brainstem to the CG-SMG before crossing to innervate contralateral lobes. This peripheral arrangement suggests that spatially organized control can be implemented downstream of peripheral ganglia, representing a potential evolutionary adaptation. More broadly, it calls for re-examination of neural laterality paradigms and identifies peripheral ganglia as decision nodes and therapeutic targets for organ-specific neuromodulation.

Despite these advances, several limitations warrant mention. First, the specific neuronal subtypes within the LPGi that mediate contralateral projections remain unidentified. Future research employing genetic labeling strategies and single-cell RNA sequencing will be necessary to characterize their molecular and functional properties. Second, technical constraints prevented simultaneous bilateral viral tracing and whole-mount mouse imaging in the same animal, restricting direct assessment of interhemispheric interactions. Finally, although our murine data provide new insights into lateralized autonomic control of hepatic metabolism, their translational relevance to humans remains to be established. Progress in noninvasive imaging and neural circuit mapping may ultimately enable analogous studies in clinical settings.

In conclusion, this study establishes that the brain exerts lateralized and lobe-specific control over hepatic glucose metabolism, with contralateral regulation arising from a peripheral decussation at the porta hepatis, and a compensatory mechanism maintaining glucose homeostasis after unilateral hepatic injury. Developmentally, sympathetic projections began extending into the liver during the first postnatal week, forming a mature network by week 2 that enables both baseline regulation and adaptive responses. Together, these findings extend the concept of lateralization beyond cognition, motor control, and symmetric organs, with potential implications for targeted neuromodulation strategies in organ-specific diseases.

Methods

Animals

All animal experiments were approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University and adhered to the principles outlined in the Guide for the Care and Use of Laboratory Animals. Mice were housed under a 12-hour light-dark cycle at 22 °C with ad libitum access to food and water, unless otherwise specified. The age and number of mice used in each experiment are provided in the figure legends. Littermates of the same sex were randomly assigned to either experimental or control groups.

Viral vectors

For retrograde tracing of LPGi projection to individual hepatic lobes, we injected PRV-CAG-EGFP (BrainVTA, Cat#P03001) and PRV-CAG-mRFP (BrainVTA, Cat#P03002) into distinct hepatic lobes, respectively. For chemogenetic manipulation, we used a combination of AAV9-GAD67-Cre (BrainVTA, Cat#PT-6600) with AAV9-DIO-hM3Dq (BrainVTA, Cat#PT-0019) or AAV9-DIO-mCherry (BrainVTA, Cat#PT-0115). For optogenetic manipulation, we used a combination of AAV9-GAD67-Cre with AAV9-DIO-ChR2-mCherry (BrainVTA, Cat#PT-0002) or AAV9-DIO-mCherry. For anterograde tracing from the CG-SMG to hepatic lobes, we injected AAV9-TH-Cre (BrainVTA, Cat#PT-2947) with AAV9-EF1α-DIO-Axon-EGFP (BrainVTA, Cat#BC-0197) into the unilateral CG-SMG. All viral vectors were used at titers >5 × 10¹² vg/mL.

Brain stereotactic injection

Mice were anesthetized with isoflurane (RWD Life Science Co., Ltd., Cat#68811). For brain injection, the coordinates of LPGi were determined using the Allen Mouse Brain Atlas (AP −6.75 mm, ML ±0.95 mm, DV −5.85 mm). A total of 200 nL of viral vector was bilaterally delivered at a rate of 50 nL/min.

Intrahepatic sympathetic nerve tracing

After laparotomy, the intestines were gently exteriorized and covered with a sterile saline-moistened gauze to expose the CG-SMG. For anterograde tracing of CG-SMG projections to hepatic lobes, AAV9-TH-Cre and AAV9-EF1α-DIO-Axon-EGFP were injected into left CG or right CG, and tissues were collected 3 weeks post-injection. For retrograde tracing, 5,000 nL of 1 mg/ml Alexa Fluor 647-conjugated wheat germ agglutinin (WGA 647, Thermo, Cat#W32466) and Alexa Fluor 555-conjugated wheat germ agglutinin (WGA 555, Thermo, Cat#W32464) were injected into the left- and right-sided lobes, respectively, using an infusion rate of 100 nL/min. Tissues were harvested 3 days thereafter.

Intrahepatic sympathetic denervation

For liver injections, we calculated volumes based on the proportion of each lobe relative to the total liver weight. We injected a volume of 3,000 nL of 6-hydroxydopamine (6-OHDA, 12 mg/ml; Sigma, Cat#162957) solution into each lobe at a flow rate of 1,000 nL/min.

Whole-mount clearing and imaging

The liver and trunk staining procedure was performed according to established protocols as previously described.^(14,21) In brief, the liver tissues were fixed in 4% paraformaldehyde (PFA), followed by permeabilization in PBS containing 0.2% Triton X-100, 20% DMSO, and 0.3 M glycine. Immunostaining was performed using the primary antibody against TH (Merck, Cat #AB152, 1:500), followed by incubation with a secondary antibody (Alexa Fluor 568-conjugated donkey anti-rabbit IgG, Thermo Fisher, Cat#A-10042). After staining, tissues were embedded in 1% agarose, dehydrated, and cleared using dibenzyl ether (DBE; Sigma, Cat#422053).

The brain-liver neurocircuit visualization was performed as previously described.⁽²⁵⁾ At 5 days post-injection of PRV, the mice were anesthetized with isoflurane and perfused with PBS. Samples were permeabilized in PBS containing 1.5% goat serum, 0.5% Triton X-100, 0.5 mM methyl-β-cyclodextrin, and 0.2% trans-1-acetyl-4-hydroxy-l-proline. Following permeabilization, samples were stained with mRuby3 nanobody (NanoTag, Cat#N3302-AF647-L, 1:5,000). They were then dehydrated with upgraded tetrahydrofuran (THF; Sigma, Cat#401757), and subsequently incubated in dichloromethane (Sigma, Cat#270997). Finally, samples were cleared by benzyl alcohol and benzyl benzoate mixture (Sigma, Cats #24122 and #W213802).

The cleared samples were imaged using a light-sheet fluorescence microscope (Nuohai, LS18) at 1.25×, 3.2×, and 12.6× magnification. Finally, full-tissue image stacks were reconstructed and analyzed using Imaris ×64 software (version 9.5.0, Bitplane).

Chemogenetic and optogenetic manipulations

For chemogenetic manipulation, we stereotaxically injected mice with 200 nL of AAV carrying the Cre-dependent activator DREADD (AAV9-DIO-hM3Dq-mCherry) and allowed them to recover for at least four weeks before conducting tests.

For optogenetic manipulation, mice received stereotaxic injections of 200 nL AAV9-DIO-ChR2-mCherry into the LPGi, followed by implantation of optical fibers (Inper, Cat#D3429). After a four-week recovery, mice were habituated to a fiber-optic patch cord in the home cage for 2 hours before the stimulation. Blue light (465 nm) stimulation, consisting of pulse trains (10 ms pulses of 20 Hz at approximately 10 mW; 1 s on, 1 s off), was delivered.

Biochemical assays

The concentrations of epinephrine (EPI) and norepinephrine (NE) were quantified using the EPI enzyme-linked immunosorbent assay (ELISA) kit (Kexing, Cat#Fankew F2351-A) and NE ELISA kit (Kexing, Cat#Fankew F2533-A). PEPCK activity assay kit (Kexing, Cat#Fankew F2533) and G6PC activity assay kit (Kexing, Cat#Fankew F2736-A) were used to quantify the activity of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6PC).

Western blot analysis

Tissue samples were homogenized in RIPA lysis buffer (Beyotime, Cat#P0013B) with 100 µM protease and phosphate inhibitors (Beyotime, Cat#P1045) using a homogenizer (JieChen Instrument, JCZYM-48R) at 4 °C. The following primary antibodies were used: PEPCK (Santa, Cat#sc-271029, 1:1,000), G6PC (Novus, Cat#NBP1-80533, 1:1,000), pPYGL (Ser15) (Abcam, Cat#ab227043, 1:1,000), PYGL (Proteintech, Cat#15851-1-AP, 1:1,000), pGS (Ser641) (Cell Signaling Technology, Cat#3891S, 1:1,000), GS (Santa, Cat#sc-81173, 1:1,000), TH (Merck, Cat#AB152, 1:1,000), and α-Tubulin (Huabio, Cat#ER130905, 1:10,000), followed by incubation with appropriate secondary antibodies. Imaging of membranes was performed with the iBright Imaging Systems (Thermo, iBright 1500). We quantified the signal intensities by performing densitometric analyses using Fiji (ImageJ, NIH).

Histology and immunofluorescence analyses

Mouse livers and brains were collected separately and fixed with 4% PFA overnight at 4 °C. For Periodic Acid-Schiff (PAS) staining, 25 µm paraffin sections were prepared and stained with a PAS staining kit (Beyotime, Cat#C0142S). For immunostaining, 25-µm-thick frozen sections were prepared by a freezing microtome (Leica, CM 1950) and stained with primary antibodies, including anti-c-FOS (Synaptic Systems, Cat#226-008, 1:1,000), anti-TH (Merck, Cat#AB152, 1:500), anti-pPYGL (Ser15) (Abcam, Cat#ab227043, 1:100), and anti-pGS (Ser641) (Cell Signaling Technology, Cat#3891S, 1:100), followed by incubation with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit IgG, Yeasen, Cat#33116ES60). The sections were imaged using confocal microscopes (Zeiss, LSM 700). Images were quantified with ImageJ (v1.8.0).

Quantification and statistical analysis

An unpaired two-tailed Student’s t-test was used to compare differences between two groups. Comparisons across multiple groups were conducted using one-way ANOVA with the Tukey test. All results, including graphs, were expressed as means ± SEM unless otherwise specified. Significance was defined as p < 0.05, with significance annotations of *p < 0.05, **p < 0.01, ***p < 0.001; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001. Statistical analyses were conducted using software such as GraphPad Prism (v.10.0) and ImageJ (v1.8.0).

Supplementary evidence for contralateral projections from the brain to the liver, related to Figure 1.
(A) Schematic illustration of hepatic lobes with anatomical labeling. (B) Represent images of hepatic lobes after PRV-EGFP injections. Scale bars, 150 μm. (C-F) Representative immunofluorescence images of PRV-labeled neurons (EGFP) in the left (top) and right (bottom) LPGi following PRV injections into left lateral lobe (C), right posterior lobe (D), caudate lobe (E), and porta hepatis (F). Scale bars, 100 μm.

Chemogenetic activation of LPGi induces c-Fos expression, promotes hepatic glycogenolysis, and activates glycogenolytic enzymes, related to Figure 2.
(A) Representative immunofluorescence images of c-FOS⁺ LPGi neurons in hM3Dq mice following 1 h stimulation (Left), with quantification of c-FOS⁺ neuron percentage (right, n = 3). Scale bars, 100 μm. (B) Representative PAS staining images of median (top) and right anterior (bottom) lobes in hM3Dq mice following 1 h stimulation, with semi-quantitative analysis of glycogen levels (right, n = 5). Scale bars, 100 µm. (C and D) Western blot analysis of PEPCK and G6PC proteins in the median (C, top) and right anterior (D, top) lobes, with densitometric quantified (bottom, n = 3). (E and F) Enzyme activity of PEPCK and G6PC in the median (E) and right anterior (F) lobes (n = 3). Data are expressed as means ± SEM, with individual values shown for all experiments. *p < 0.05, ***p < 0.001; ^##p < 0.01, ^###p < 0.001; ^$$$p < 0.001. A one-way ANOVA with the Tukey test was used for (A-F).

Optogenetic activation of LPGi confirms contralateral lobe-specific mobilization observed in Figure 2.
(A) Scheme for injecting AAV-DIO-ChR2 or AAV-DIO-mCherry (top). Blood glucose levels following optogenetic activation of left, right, or bilateral LPGi, measured at −10, 30, 60, 90, and 120 min (bottom, n = 6) (B and C) Representative immunofluorescence images of pGS (B) and pPYGL (C) expression in median (top) and right anterior (bottom) lobes after 1 h optogenetic activation of left, right, or bilateral LPGi, with quantification of relative fluorescence intensity (n = 5). Scale bars, 100 μm. (C) Western blot analysis of pPYGL, PYGL, pGS, and GS in median (left) and right anterior (right) lobes after 1 h activation, with densitometric quantification (bottom, n = 3). (D) Plasma concentrations of norepinephrine (NE) (left, n = 5) and epinephrine (EPI) (right, n = 5) following 1 h optogenetic activation. (E) NE contents in median (left, n = 5) and right anterior (right, n = 5) lobes after 1 h optogenetic activation. Data are expressed as means ± SEM, with individual values shown for all experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; ^$p < 0.05, ^$$$p < 0.001. A one-way ANOVA with the Tukey test was used for (A -F).

Optogenetic activation of LPGi recapitulates c-Fos expression, hepatic glycogenolysis, and activity of glycogenolysis-related enzymes observed in Figure S2.
(A) Representative immunofluorescence images of c-FOS⁺ neurons in LPGi after 1 h optogenetic stimulation, with quantification of ratio of c-FOS⁺ neurons (right, n = 3). Scale bars, 100 μm. (B) Representative PAS staining images of the median (top) and right anterior lobes (bottom) lobes following 1 h optogenetic stimulation, with semi-quantitative analysis of glycogen levels (n = 5). Scale bars, 100 µm. (C and D) Western blot analysis of PEPCK and G6PC proteins in median (C) and right anterior( D) lobes, with densitometric quantification (n = 3). (E and F) Enzyme activity of PEPCK and G6PC in median (E) and right anterior lobes (F) (n = 3). Data are expressed as means ± SEM, with individual values shown for all experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; ^$$$p < 0.001. A one-way ANOVA with the Tukey test was used for (A-F).

Validation of sympathetic nerve ablation, enhanced glycogenolysis in non-denervated hepatic lobes, and accurate targeting of AAV and WGA injections, related to Figure 3 and 4.
(A) Densitometric quantification of TH protein in median and right anterior lobes after denervating left- (left) or right-sided (right) lobes. (B-E) Representative PAS staining images of median (top) and right anterior (bottom) lobes in left- (B) and right-sided (D) lobes denervated mice, with semi-quantitative analysis of glycogen levels (C and E, n = 5). Scale bars, 100 µm. (A) Represent slices of the CG in unilateral AAV-TH-axon-EGFP injected mice. Scale bars, 20 μm. (B) Represent slices of hepatic lobes after injecting WGA 647 and WGA 555 into left- and right-sided lobes. Scale bars, 50 μm. Data are expressed as means ± SEM, with individual values shown for all experiments. ***p < 0.001; Unpaired Student’s t-test for (A, C, and E).

Data availability

All data generated or analyzed during this study are included in themanuscript and supporting files. Source data files have been provided for all figures.

Acknowledgements

We thank the following agencies for financial support: the National Natural Science Foundation of China (82301436, 82371311, 81930109, and 82321005), National Key R&D Program of China (2021YFA1301300, 2023YFA1801900); the Natural Science Foundation of Jiangsu Province (BK20221050, BG2024045), the Overseas Expertise Introduction Project for Discipline Innovation (G20582017001), the Jiangsu Specially Appointed Professor Program, and the International Postdoctoral Talent Recruitment Program, the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX25_1076).

Additional information

Author contribution

H.X., H.H., and X.Z. conceived, designed, and supervised the project. Z.W., M.R., Y.C., and Y.L. performed glucose level monitoring and data analysis. X.G., K.W., Y.C., and L.J. performed chemogenetic and optogenetic experiments. X.S., S.W., and J.W. performed whole-mount immunolabeling. H.X. and H.H. wrote the manuscript with input from H.W. and X.C. All authors validated and approved the final manuscript.

Ethics approval and consent to participate

Abbreviations

AUC: area under the curve
CG: celiac ganglion
CNS: central nervous system
ChR2: channelrhodopsin-2
ELISA: enzyme-linked immunosorbent assay;
EPI: epinephrine
G6PC: glucose-6-phosphatase
HPA: hypothalamic-pituitary-adrenal
LPGi: lateral paragigantocellular nucleus
NE: norepinephrine
6-OHDA: 6-hydroxydopamine
PAS: periodic acid-Schiff
PEPCK: phosphoenolpyruvate carboxykinase
PRV: pseudorabies virus
GS: glycogen synthase
PYGL: glycogen phosphorylase
WGA: wheat germ agglutinin.

Funding

MOST | National Natural Science Foundation of China (NSFC) (82301436)

Hao Xie

MOST | National Natural Science Foundation of China (NSFC) (82371311)

Hao Xie

MOST | National Natural Science Foundation of China (NSFC) (81930109)

Haiping Hao

MOST | National Natural Science Foundation of China (NSFC) (82321005)

Haiping Hao

MOST | National Key Research and Development Program of China (NKPs) (2021YFA1301300)

Haiping Hao

MOST | National Key Research and Development Program of China (NKPs) (2023YFA1801900)

Hao Xie

JST | Natural Science Foundation of Jiangsu Province (Jiangsu Natural Science Foundation) (BK20221050)

Hao Xie

JST | Natural Science Foundation of Jiangsu Province (Jiangsu Natural Science Foundation) (BG2024045)

Haiping Hao

111 Plan | Overseas Expertise Introduction Project for Discipline Innovation (G20582017001)

Haiping Hao

Jiangsu Specially Appointed Professor Program, and the International Postdoctoral Talent Recruitment Program, the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX25_1076)

Hao Xie

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Article and author information

Author information

Zhonglong Wang
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
- These authors contributed equally: Zhonglong Wang, Xiangfei Gong, Li Jiang, and Ke Wang
Xiangfei Gong
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
- These authors contributed equally: Zhonglong Wang, Xiangfei Gong, Li Jiang, and Ke Wang
Li Jiang
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
- These authors contributed equally: Zhonglong Wang, Xiangfei Gong, Li Jiang, and Ke Wang
Ke Wang
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
- These authors contributed equally: Zhonglong Wang, Xiangfei Gong, Li Jiang, and Ke Wang
Xinyuan Sun
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
Yingxi Li
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
Mengtong Ran
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
Yanshen Chen
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
Hongdong Wang
Department of Endocrinology, Endocrine and Metabolic Disease Medical Center, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
Xuehui Chu
Department of Pancreatic and Metabolic Surgery, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
Shun Wang
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
Junjie Wang
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
Xiao Zheng
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
- For correspondence: xiaozheng@cpu.edu.cn
Haiping Hao
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
ORCID iD: 0000-0003-2522-7546
- For correspondence: haipinghao@cpu.edu.cn
Hao Xie
College of Pharmacy, China Pharmaceutical University, Nanjing, China, State Key Laboratory of Natural Medicines, Institute of innovative Drug Discovery and Development, Jiangsu Provincial Key Laboratory of Targetome and Innovative Drugs, China Pharmaceutical University, Nanjing, China
ORCID iD: 0009-0009-4778-2912
- For correspondence: haoxie@cpu.edu.cn

Author Notes

Competing interests: No competing interests declared

Version history

Preprint posted: October 27, 2025
Sent for peer review: October 27, 2025
Reviewed Preprint version 1: January 12, 2026

Cite all versions

You can cite all versions using the DOI https://doi.org/10.7554/eLife.109599. This DOI represents all versions, and will always resolve to the latest one.

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Significance of findings

Strength of evidence

Abstract

Highlights

Introduction

Results

1. Contralateral projections from the LPGi to distinct hepatic lobes

The brain contralaterally projects to the liver.

2. Differential HGP by LPGi lateralization

LPGi stimulation raises blood glucose by driving contralateral, lobe-specific hepatic glycogenolysis.

3. Compensatory neural responses following unilateral hepatic denervation

LPGi maintains glucose balance through compensatory use of ipsilateral hepatic lobes under contralateral lobe impairment.

4. Peripheral decussation of sympathetic hepatic control at the porta hepatis

Sympathetic projections to the liver decussate at the porta hepatis, progressively formed during development.

Discussion

Methods

Animals

Viral vectors

Brain stereotactic injection

Intrahepatic sympathetic nerve tracing

Intrahepatic sympathetic denervation

Whole-mount clearing and imaging

Chemogenetic and optogenetic manipulations

Biochemical assays

Western blot analysis

Histology and immunofluorescence analyses

Quantification and statistical analysis

Supplementary evidence for contralateral projections from the brain to the liver, related to Figure 1.

Chemogenetic activation of LPGi induces c-Fos expression, promotes hepatic glycogenolysis, and activates glycogenolytic enzymes, related to Figure 2.

Optogenetic activation of LPGi confirms contralateral lobe-specific mobilization observed in Figure 2.

Optogenetic activation of LPGi recapitulates c-Fos expression, hepatic glycogenolysis, and activity of glycogenolysis-related enzymes observed in Figure S2.

Validation of sympathetic nerve ablation, enhanced glycogenolysis in non-denervated hepatic lobes, and accurate targeting of AAV and WGA injections, related to Figure 3 and 4.

Data availability

Acknowledgements

Additional information

Author contribution

Ethics approval and consent to participate

Abbreviations

Funding

References

Article and author information

Author information

Zhonglong Wang*

Xiangfei Gong*

Li Jiang*

Ke Wang*

Xinyuan Sun

Yingxi Li

Mengtong Ran

Yanshen Chen

Hongdong Wang

Xuehui Chu

Shun Wang

Junjie Wang

Xiao Zheng

Haiping Hao

Hao Xie

Author Notes

Version history

Cite all versions

Copyright

Metrics

Zhonglong Wang

Xiangfei Gong

Li Jiang

Ke Wang