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The development of hepatic steatosis depends on the presence of liver-innervating parasympathetic cholinergic neurons in mice fed a high-fat diet [1]

['Jiyeon Hwang', 'The Fleischer Institute For Diabetes', 'Metabolism', 'Albert Einstein College Of Medicine', 'Bronx', 'New York', 'United States Of America', 'Division Of Endocrinology', 'Department Of Medicine', 'Junichi Okada']

Date: 2024-10

Hepatic lipid metabolism is regulated by the autonomic nervous system of the liver, with the sympathetic innervation being extensively studied, while the parasympathetic efferent innervation is less understood despite its potential importance. In this study, we investigate the consequences of disrupted brain–liver communication on hepatic lipid metabolism in mice exposed to obesogenic conditions. We found that a subset of hepatocytes and cholangiocytes are innervated by parasympathetic nerve terminals originating from the dorsal motor nucleus of the vagus. The elimination of the brain–liver axis by deleting parasympathetic cholinergic neurons innervating the liver prevents hepatic steatosis and promotes browning of inguinal white adipose tissue (ingWAT). The loss of liver-innervating cholinergic neurons increases hepatic Cyp7b1 expression and fasting serum bile acid levels. Furthermore, knockdown of the G protein-coupled bile acid receptor 1 gene in ingWAT reverses the beneficial effects of the loss of liver-innervating cholinergic neurons, leading to the reappearance of hepatic steatosis. Deleting liver-innervating cholinergic neurons has a small but significant effect on body weight, which is accompanied by an increase in energy expenditure. Taken together, these data suggest that targeting the parasympathetic cholinergic innervation of the liver is a potential therapeutic approach for enhancing hepatic lipid metabolism in obesity and diabetes.

Funding: This work was supported by the NIH Blueprint for Neuroscience Research (AT011653 to Y.H.J), NIH (TR003313 to Y.-H.J), and NIDDK (DK092246 to Y.-H.J and G.J.S., DK020541 to Y.-H.J, G.J.S., and J.E.P. and DK110063 to J.E.P.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we aimed to investigate the impact of the loss of the parasympathetic brain–liver cholinergic axis on hepatic lipid metabolism in mice fed a high-fat diet (HFD). Our anterograde and retrograde neuronal mapping and 3D imaging of cleared liver tissues revealed that a subset of hepatocytes and cholangiocytes received vAChT-positive nerve terminals from the DMV. The removal of the liver-innervating parasympathetic cholinergic neurons prevented the development of hepatic steatosis. This decrease in hepatic fat accumulation was linked to the beiging of ingWAT caused by activation of the G protein-coupled bile acid receptor 1 (GPBAR1 or TGR5) in ingWAT.

Dynamic crosstalk between the liver and white adipose tissue (WAT) appears essential for hepatic fat accumulation and the development of MAFLD [ 9 ]. VLDL-TG synthesized in the liver is secreted and transported to adipose tissue for storage in the postprandial state [ 10 ]. Fasting increases lipolysis in adipocytes, and the free fatty acids (FFAs) released from WAT are transported to the liver for energy metabolism [ 10 ]. In human studies, most fatty acids constituting VLDL-TG are derived from adipose tissue [ 11 ]. However, in obesity, adipose tissue can no longer effectively store lipids, thus redirecting them toward other organs, primarily the liver [ 9 ]. Interestingly, the liver has the capability to control lipid uptake and lipolysis in WAT by activating bile acid (BA) signaling pathways [ 12 ]. Vagotomized rats exhibit reduced expression of the cholesterol 7 alpha-hydroxylase (Cyp7a1) gene, the rate-limiting enzyme in the classic bile acid synthesis pathway in the liver, while displaying increased bile acid levels in the serum and bile, in part via enhanced intestinal passive absorption of BAs [ 13 ]. Biliary epithelial cells in rats express the muscarinic acetylcholine receptor type 1, and its activation by the cholinergic agonist carbachol reduces bile flow [ 14 ]. These results suggest that the parasympathetic cholinergic innervation to the liver has the potential to regulate bile acid synthesis and outflow from the liver, which would subsequently result in a decrease in lipolysis within the WAT. In fact, a recent 3D/Airyscan imaging study has demonstrated vesicular acetylcholine transporter (vAChT)-positive parasympathetic nerve terminals on the bile duct in human liver tissues [ 15 ].

Metabolic dysfunction-associated fatty liver disease (MAFLD), particularly its progressive form known as metabolic dysfunction-associated steatohepatitis (MASH), is now the leading cause of liver transplantation in Western countries [ 1 ]. MAFLD is characterized by the accumulation of excessive amounts of fat in the liver, which is determined by multiple factors including the uptake of fatty acids by hepatocytes, de novo lipogenesis (DNL), mitochondrial and peroxisomal fatty acid oxidation, and lipid export from the liver [ 2 , 3 ]. Results from recent studies support important roles for the autonomic nervous system in mediating multiple critical hepatic lipid metabolism pathways. For example, MAFLD both in mice and humans [ 4 ] is associated with significant plasticity in the sympathetic innervation to the liver, including swollen axonal varicosities, nerve fiber retraction, and abnormal axonal sprouting. The vagus nerve also exerts a significant parasympathetic influence on the regulation of hepatic lipid metabolism. For example, activation of central leptin receptors reduces hepatic fat accumulation, which requires surgically intact hepatic vagal innervation in lean and high-fat fed rodents [ 5 ]. Similarly, the anti-steatotic effect of leptin is dependent on vagal innervation in humans [ 6 ]. In addition, central infusion of oleic acid suppresses triglyceride-rich very low-density lipoproteins (VLDL-TG) secretion through hepatic vagal innervation in rats [ 7 ]. The hepatic vagus nerve regulates the development of MAFLD via altering hepatic inflammation [ 8 ]. However, a major challenge in interpreting the results of hepatic vagal surgical denervation studies is that it interrupts both vagal sensory and parasympathetic cholinergic nerve fibers, which pass through the hepatic vagus nerve. Consequently, the specific role for parasympathetic cholinergic innervation of the liver plays in regulating hepatic lipid metabolism remain unclear.

Intriguingly, a growing number of studies have revealed sex-specific differences in metabolic homeostasis, diabetes, and obesity in both rodents and humans [ 43 – 49 ]. For instance, premenopausal women were relatively protected from diseases associated with metabolic syndrome [ 50 ]. Female rodents were less prone to developing diet-induced obesity (DIO), insulin resistance, hyperglycemia, and hypertriglyceridemia [ 43 , 44 , 47 , 48 ]. In particular, C57BL/6 female mice were less likely to develop DIO and hyperglycemia than C57BL/6 male mice during high-fat feeding [ 47 – 49 , 51 , 52 ]. Our study also found that control female mice did not develop DIO or hyperglycemia ( S10A–S10F Fig ), and hepatic cholinergic loss of function had no effect on body weight or glucose homeostasis ( S10A–S10F Fig ). In other words, both groups did not show any notable differences in body weight, food intake, body composition, blood glucose levels, GTT, and ITT compared with the controls ( S10A–S10H Fig ).

To evaluate whether ChAT Cre mice without parasympathetic cholinergic neurons innervating the liver exhibit any changes in their ability to dispose a glucose load, we conducted intraperitoneal glucose tolerance tests (i.p.GTT) and found no significant difference in blood glucose levels in response to a bolus of glucose (i.p., 2 g/kg) between the groups ( S9C and S9D Fig ). However, the experimental mice showed increased insulin sensitivity ( S9E Fig ). During the insulin tolerance tests (ITTs) (i.p., 1 U/kg), blood glucose levels were significantly lower in ChAT Cre mice injected with AAVrg-FLEX-dtA than in controls ( S9E Fig ). Furthermore, the improved insulin tolerance in the experimental group was associated with reduced plasma insulin levels relative to those in the controls ( S9F Fig ). These results indicate that the loss of DMV cholinergic input to the liver enhanced insulin sensitivity in ChAT Cre male mice fed HFD.

We evaluated whether deleting the brain–liver axis affects glucose homeostasis in mice fed an HFD, as the liver plays a crucial role in regulating systemic glucose levels [ 42 ]. To assess glucose intolerance and insulin sensitivity, we measured blood glucose and insulin levels. Our results showed that basal glucose levels were lower in ChAT Cre mice receiving AAVrg-FLEX-dtA injection than in the controls ( S9A Fig ). Additionally, the loss of DMV cholinergic neurons innervating the liver led to a significant reduction in fasting glucose levels ( S9B Fig ).

The reduction in body weight, despite no change in energy intake, may be due to an increase in energy expenditure. To assess metabolic rate, we placed animals in metabolic cages and observed that the experimental mice consumed more oxygen and produced more carbon dioxide during the dark phase than the controls ( S8E and S8F Fig ). These findings indicate an increase in total energy expenditure in the experimental mice ( S8G Fig ). However, the respiratory exchange ratio was similar between the 2 groups ( S8H Fig ). Notably, the experimental mice exhibited more locomotor activity during the dark phase ( S8I and S8J Fig ). Collectively, these results suggest that deleting liver-projecting cholinergic neurons plays a role in increasing energy expenditure.

We also investigated whether the disruption of the brain–liver axis could impact body weight in mice fed HFD. After 10 weeks of HFD feeding, we observed a small but significant difference in body weight gain between the control and experimental groups ( S8A Fig ). The experimental mice gained less weight than the control mice during the HFD period. This decrease in weight gain was closely related to a significant reduction in fat mass in the experimental group, while lean mass was similar between the 2 groups ( S8B Fig ). When we compared adipose tissue mass collected from controls with that of adipose tissue from the experimental group, we found a significant decrease in the mass of ingWAT ( S8C Fig ). It is important to note that both groups consumed an equal amount of food during the light and dark phases ( S8D Fig ).

(A) Gel images showing the western blot analysis of ingWAT from experimental mice revealed an increase in the expression of pS660-HSL when compared to the control group. (B) Graph showing plasma glycerol levels in the controls (black circle, n = 6 mice) and the experimental groups (blue circle, n = 5 mice). Unpaired t test, **p < 0.01. (C, D) Representative images of liver sections stained with HE (C) and Oil Red O (D). Upper panel: scale bar, 100 μm; bottom panel: higher magnification view of the black dotted square scale bar, 50 μm. (E) Graph showing hepatic TG levels in the controls (n = 5 mice) and the experimental groups (n = 6 mice). Unpaired t test, *p < 0.05. The data supporting the graphs shown in the figure (Fig 7B and 7E) are available in the S1 Data file. AAV, adeno-associated virus; HE, hematoxylin and eosin; HFD, high-fat diet; TG, triglyceride.

We examined whether Tgr5 knockdown impacts HSL-mediated lipolysis in ingWAT. Our findings showed an increase in phospho-HSL levels in the ingWAT ( Fig 7A ), which subsequently led to higher levels of plasma glycerol ( Fig 7B ). Considering the well-established connection between elevated plasma glycerol levels and lipid accumulation in the liver, we sought to determine if Tgr5 knockdown results in hepatic steatosis. Our results revealed the existence of large lipid droplets and ballooned hepatocytes in the liver sections of mice that received Tgr5 siRNA ( Fig 7C ). Furthermore, Oil Red O staining confirmed the accumulation of lipids in the livers of mice treated with Tgr5 siRNA in the ingWAT ( Fig 7D ). Additionally, we found that the knockdown of the Tgr5 gene in the ingWAT of ChAT Cre mice without DMV cholinergic neurons to the liver resulted in a significant increase in hepatic TG content, which is indicative of enhanced lipid accumulation in the liver ( Fig 7E ). Collectively, our findings support the idea that cross-talk between the liver and ingWAT through elevated bile acid signaling plays a vital role in maintaining the lipid metabolism balance in both the liver and ingWAT.

(A, B) Graphs showing mRNA and protein expression levels of Tgr5 in ingWAT. There was a significant increase in TGR5 expression in the ingWAT of ChAT Cre mice that received AAVrg-FLEX-dtA (RT-qPCR, control (open circle), n = 7 mice; experimental (closed circle), n = 7 mice, unpaired t test, **p < 0.01; western blotting, control (open circle), n = 3 mice; experimental (closed circle), n = 3 mice, unpaired t test, ***p < 0.001). (C) Schematic illustration showing our experimental configurations. (D) Reduction in Tgr5 gene and protein levels in ChAT Cre mice following administration of AAVrg-FLEX-dtA to the liver (black circle) and another injection of Tgr5 siRNA into both sides of the ingWAT (blue circle) (unpaired t test, *** P < 0.001). (E) Representative images of ingWAT sections stained with HE. ChAT Cre mice without DMV cholinergic neurons innervating the liver receiving an injection of Tgr5 siRNA into ingWAT exhibited larger adipocytes compared to the control mice (upper panel). Scale bar, 50 μm. Bottom panel: higher magnification view of the black dotted square. Arrow represents crown-like structure. Scale bar, 100 μm. (F, G) Plot showing the distribution of average adipocyte size. The mean cell area was significantly larger in the experimental group receiving Tgr5 siRNA than in the experimental group receiving control siRNA (I) (control, n = 5 mice; experimental, n = 5 mice, unpaired t test, *p < 0.05). The data supporting the graphs shown in the figure (Fig 6A, 6B, 6D, 6F, and 6G) are available in the S1 Data file. AAV, adeno-associated virus; BA, bile acid; HE, hematoxylin and eosin; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

We then examined whether the expression of its corresponding receptor, TGR5, is altered in the ingWAT of the experimental mice. Our observations revealed elevated levels of both TGR5 gene and protein expression in the ingWAT of the experimental mice compared to the control group ( Fig 6A and 6B ). This suggests that the increase in serum BAs and Tgr5 expression in the ingWAT leads to beiging of the ingWAT in the experimental mice. To investigate this possibility, we administered Tgr5 siRNA or control siRNA into the ingWAT of ChAT Cre mice without liver-innervating cholinergic neurons to exclusively knock down Tgr5 in the ingWAT ( Fig 6C ). Ten weeks after a viral infection, we conducted RT-qPCR and western blot analyses on samples and found that the Tgr5 gene and protein levels in ingWAT were significantly reduced following the injection of Tgr5 siRNA ( Fig 6D ), demonstrating the effectiveness of our approach in knocking down Tgr5 expression. Our experiments also revealed that the number of large-sized adipocytes and crown-like structures in the ingWAT of mice that received Tgr5 siRNA was increased ( Fig 6E ), accompanied by a notable increase in adipocyte area ( Fig 6F and 6G ).

(A) Graph showing mRNA expression of the enzymes involved in BA synthesis in liver. While there was no difference in Cyp7a1 gene expression, the loss of hepatic cholinergic input significantly up-regulated Cyp7b1 mRNA expression (control (open circle), n = 7–8 mice; experimental (closed black circle), n = 7 mice). (B) Graphs showing Cyp7a1 and Cyp7b1 gene expression in the periportal (PP), midlobular (Mid), and pericentral (PC) zones in the livers of the controls and the experimental groups. (C) Graph showing the fasting serum BA levels in the control group (n = 6 mice) and the experimental group (n = 5 mice). Unpaired t test, *p < 0.05. (D) Graph showing fasting serum BA profile in the control (n = 4 mice) and experimental (n = 4 mice) mice. The Mann–Whitney test revealed a statistically significant difference between the 2 groups (*p < 0.05). The data supporting the graphs shown in the figure (Fig 5A–5D) are available in the S1 Data file. AAV, adeno-associated virus; BA, bile acid.

How would removing hepatic cholinergic innervation drive these reductions in adiposity in the ingWAT, which does not receive parasympathetic cholinergic innervation in rodents [ 38 , 39 ]. Our results suggest that removing hepatic parasympathetic cholinergic input enhances liver-adipose crosstalk, limiting ingWAT lipolysis and thereby limiting hepatic steatosis during HFD feeding. Previous studies have indicated that bile acid and its corresponding receptor TGR5 are crucial in the browning of WAT [ 12 , 40 ]. We thus investigated whether the loss of function of liver-innervating cholinergic neurons affects the expression of genes related to bile acid synthesis, such as Cyp7a1 and Cyp7b1. We found that hepatic cholinergic loss of function up-regulated expression of the Cyp7b1 gene involved in an alternative BA synthesis pathway [ 41 ] ( Fig 5A ). Furthermore, our spatial transcriptomic analysis of the liver revealed that ChAT Cre mice without DMV cholinergic neurons innervating the liver displayed a trend toward an increase in Cyp7b1 gene expression in all 3 zones ( Fig 5B ), whereas Cyp7a1 expression was not altered, as observed in qPCR experiments ( Fig 5A and 5B ). Elevated Cpy7b1 expression led to a substantial increase in fasting serum BA levels in ChAT Cre mice that received AAVrg-FLEX-dtA ( Fig 5C ). In our further analysis of the fasting serum BA profiles in both control and experimental mice, we observed a significant increase in primary BAs such as cholate and chenodeoxycholate, which was attributed to the loss of hepatic cholinergic input ( Fig 5D ).

WAT lipolysis in obesity can result in an excess of fatty acids and glycerol in the liver [ 36 ]. This lipid influx can cause hepatic TG synthesis, which results in MAFLD [ 37 ]. We investigated whether eliminating cholinergic input to the liver from the DMV would decrease HSL-mediated lipolysis in ingWAT. The western blot analysis of ingWAT demonstrated a decrease in the levels of phospho-HSL in the experimental mice ( Fig 4I ). Moreover, we observed a decrease in plasma glycerol levels in the experimental mice compared to the control mice ( Fig 4J ). Taken together, these findings demonstrate that the loss of hepatic cholinergic innervation led to an increase in the expression of thermogenic genes while simultaneously reducing lipolysis in ingWAT.

RT-qPCR analysis revealed that deleting DMV cholinergic neurons innervating the liver significantly up-regulated the expression of Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha (PGC1a) in ingWAT, which is required for the expression of mitochondrial biogenesis and thermogenic genes in brown fat cells [ 33 , 34 ] ( Fig 4E ). Likewise, the expression of PR (PRD1-BF1-RIZ1 homologous)-domain containing 16 (PRDM16), a transcription factor that plays a key role in browning and thermogenesis of subcutaneous WAT [ 35 ], was significantly increased in experimental mice relative to controls ( Fig 4E ). The up-regulation of these genes was associated with a robust increase in the mRNA expression of uncoupling protein 1 (Ucp1), iodothyronine deiodinase 2 (dio2), and cell death-inducing DFFA-like effector A (Cidea) ( Fig 4E ). Western blotting and immunostaining further showed an increase in UCP1 protein levels in ingWAT of ChAT Cre mice receiving AAVrg-FLEX-dtA to the liver ( Fig 4F and 4G ). In addition, daily measurement of ingWAT temperature revealed a significant elevation in the ingWAT temperature of the experimental mice in the dark phase ( Fig 4H ). These data support that a loss of parasympathetic cholinergic neurons innervating the liver induces beiging of ingWAT, resulting in an increase in thermogenesis.

(A) Representative images of the ingWAT of control and experimental mice. (B) Representative images of adipocytes in the ingWAT of control and experimental groups. Scale bar, 100 μm. (C, D) Plots showing the distribution of the average adipocyte size (left panel). Right panel: The mean cell area was significantly smaller in the experimental groups than in controls (control, n = 4 mice; experimental, n = 5 mice, unpaired t test, *p < 0.05). (E) Summary graph showing changes in thermogenic gene expression in ingWAT of ChAT Cre mice with and without DMV cholinergic neurons innervating the liver. Ablation of liver-projecting cholinergic neurons significantly up-regulated the thermogenic gene expression (unpaired t test, *p < 0.05, **p < 0.01). (F, G) Western blot images of ingWAT showing an increase in UCP1 expression in experimental mice. Images of confocal fluorescence microscopy showing increased UCP1 expression in the ingWAT of experimental mice (G). Scale bar, 50 μm. (H) Summary plots showing ingWAT temperature in the light and dark phases (control, n = 5 mice; experimental, n = 4 mice, unpaired t test, **p < 0.01). (I) Western blot images showing a decrease in pS660-HSL expression in ingWAT of experimental mice relative to controls. (J) Summary plot showing plasma glycerol levels in the control (n = 8 mice) and experimental mice (n = 8 mice, unpaired t test, *p < 0.05). The data supporting the graphs shown in the figure (Fig 4C, 4D, 4E, 4H, and 4J) are available in the S1 Data file. AAV, adeno-associated virus.

One of the hallmarks of diet-induced obeisty is a set of increases in inguinal WAT depot size, accompanied by increased adipocyte cell size [ 32 ]. Importantly, we found that ChAT Cre mice without liver-innervating cholinergic neurons had reduced ingWAT size relative to control mice ( Fig 4A ). Histological analysis of ingWAT revealed smaller adipocyes and decrease adipocyte area in experimental mice compared to the control mice ( Fig 4B–4D ). These results suggest that a loss of DMV cholinergic input to the liver prevents ingWAT hypertrophy.

A selective gene ontology (GO) analysis of the DEGs in each zonation using Enrichr-KG [ 30 ] identified the top 5 enriched GO terms in biological process ( S7D Fig ). These included “cotranslational protein targeting to membrane” (GO:0006613), “SRP-dependent cotranslational protein targeting to membrane” (GO:0006614), “protein targeting to ER” (GO:0045047), “cytoplasmic translation” (GO:0002181), and “cellular protein metabolic process” (GO:0044267). According to the mammalian phenotype ontology analysis [ 31 ], each zonation shared 3 terms, including “abnormal lipid homeostasis” (MP:0002118), “abnormal liver physiology” (MP:0000609), and “hepatic steatosis” (MP:0002628). These results further support that liver-projecting cholinergic neurons play an important role on developing hepatic steatosis in mice fed HFD.

The male control mice displayed dense lipid accumulation in the pericentral areas compared to the experimental mice. Therefore, we further examined if there are distinct gene expression patterns throughout the hepatic zonation in both groups by performing spatial transcriptomics ( S7 Fig ). Pericentral, midlobular, and periportal clusters were identified based on known zonation markers ( S7A Fig ). We found that the midlobular areas exhibited 391 differentially expressed genes (DEGs) ( S7B Fig and S1 Table ) and that 271 genes in the PC and 550 genes in the PP areas were significantly different between the groups ( S7B Fig and S2 and S3 Tables). Importantly, the loss of liver-projecting cholinergic neurons down-regulated hepatic lipogenesis-related genes, including Scd1, Fabp4, Cd36, Lpin1, Nampt, Nr1l2, and Me1 and up-regulated the Trib1 gene across the liver zonation ( S7C Fig ).

Hepatic vagal denervation results in an increase in hepatic interleukin 6 (Il6) gene expression in mice, which subsequently modulates the expression of hepatic gluconeogenic enzymes [ 29 ]. In our experiments, where we selectively deleted liver-projecting cholinergic neurons, we observed a significant decrease in Il6 gene expression ( S5A Fig ). Notably, there was no significant difference in gluconeogenic gene expression between the experimental and control groups ( S5B Fig ). Furthermore, we investigated whether the loss of parasympathetic cholinergic innervation affects sympathetic input to the liver. We used immunostaining with an anti-tyrosine hydroxylase (Th) antibody and found no detectable difference in Th-positive nerve fibers ( S6A and S6B Fig ). Additionally, hepatic norepinephrine levels were similar between the groups ( S6C Fig ). Taken together, these findings suggest that the loss of parasympathetic cholinergic neurons innervating the liver prevents the development of hepatic steatosis during HFD feeding in male mice.

As there was a lack of lipid droplets in the livers of the experimental mice, we measured the hepatic and plasma triglyceride (TG) levels. Following HFD feeding, ChAT Cre mice without parasympathetic cholinergic neurons innervating the liver on high-fat feeding exhibited reduced hepatic and plasma TG levels in the liver relative to controls ( Fig 3G ). This was associated with decreased plasma TG levels ( Fig 3G ). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of liver tissues revealed a significant decrease in the expression of the lipogenic transcription factor sterol regulatory element-binding protein 1-c (Srebp-1) in the experimental mice relative to the controls. The experimental mice also exhibited down-regulation of its target genes, including acetyl-coenzyme A carboxylase 1 (Acaca), fatty acid synthase (Fasn), stearoyl-CoA desaturase-1 (Scd1) ( Fig 3H ). In addition, the ChAT Cre mice without parasympathetic cholinergic input to the liver had increased expression of peroxisome proliferator-activated receptor alpha (PPAR-α), the central transcriptional regulator of fatty acid oxidation relative the control group. As a result, there was increased carnitine palmitoyltransferase 1a (Cpt1a) and acyl-CoA dehydrogenase medium chain (Acadm) mRNA expression in the experimental mice relative to controls ( Fig 3H ). In contrast to male mice, high-fat feeding did not cause any changes in liver histology or fat accumulation in both control and experimental nhigh-fat feeding [ 26 – 28 ].

Hematoxylin and eosin (HE) staining revealed a clear difference in the macroscopic appearance of the livers of ChAT Cre mice receiving AAVrg-FLEX-tdTomato and AAVrg-FLEX-dtA. Control mice exhibited an increased number of large and small lipid droplets and ballooned hepatocytes compared to experimental mice ( Fig 3C and 3D ). Oil Red O staining further showed that ChAT Cre mice without DMV cholinergic neurons innervating the liver contained fewer lipid droplets in the liver than controls ( Fig 3E and 3F ). These results suggest that parasympathetic cholinergic input to the liver limits hepatic steatosis during HFD feeding.

(A, B) Macroscopic appearance of livers of ChAT Cre mice receiving AAVrg-FLEX-tdTomato and AAVrg-FLEX-dtA. E-Cad expression was lower in the control group than in the experimental group. Scale bar, 100 μm. (C, D) HE staining of liver tissues revealed increased numbers of lipid droplets and ballooned hepatocytes in the controls compared to those in the experimental mice (top panel). Scale bar, 100 mm. Bottom panel: Higher magnification view of liver tissue in the black dotted square. Scale bar, 30 μm. (E, F) Oil red O staining (red) of liver tissues from ChAT Cre mice with and without hepatic cholinergic input. The experimental group did not exhibit fat accumulation in the liver parenchyma during high fat feeding. Nuclear staining (blue): hematoxylin. Scale bar, 100 μm. (G) Graphs showing hepatic (n = 9 vs. 8 mice) and plasma (n = 11 vs. 12 mice) TG levels in the controls and experimental mice. There was a significant reduction in both hepatic and plasma TG levels in the experimental group relative to those in the control group (unpaired t test, **p < 0.01, ***p < 0.001). (H) Summary graphs showing changes in hepatic mRNA expression of enzymes involved in lipid metabolism. Srebp1c mRNA expression was significantly down-regulated, while Ppara mRNA expression was increased in the livers of ChAT Cre mice receiving AAVrg-FLEX-dtA relative to controls (control, n = 6–8 mice; experimental, n = 6–8 mice, unpaired t test, *p < 0.05, **p < 0.01). The data supporting the graphs shown in the figure (Fig 3G and 3H) are available in the S1 Data file. AAV, adeno-associated virus; HE, hematoxylin and eosin; HFD, high-fat diet.

We then examined whether deleting liver-innervating cholinergic neurons alters hepatic lipid metabolism during HFD feeding. To address this question, we first investigated if there are differences in the liver histology between the groups. Liver tissues were labeled with liver zonation markers such as the pericentral marker glutamine synthetase (GS) and the periportal marker E-cadherin (E-cad) [ 24 ]. The expression pattern of these zonation markers in the livers of the control group fed a standard chow diet demonstrated that GS- and E-cad-positive areas were clearly separated in the liver ( S3A Fig ). Interestingly, high-fat feeding robustly reduced E-cad expression in periportal hepatocytes in the livers of control mice compared to control mice fed a standard chow diet ( Fig 3A ), consistent with disrupted E-cad expression in chronic liver disease in mice [ 25 ]. Surprisingly, deleting the parasympathetic cholinergic input to the liver in ChAT Cre mice fed HFD restored the expression of E-cad in periportal hepatocytes ( Fig 3B ). In contrast, GS was similarly expressed in pericentral hepatocytes in both control and experimental mice fed HFD ( Fig 3A and 3B ).

(A) Schematic illustration of the experimental configurations, where AAVrg-FLEX-tdTomato (control, ο) and AAVrg-FLEX-dTA (•) viral vectors were injected directly into the livers of ChAT Cre mice. (B) Images of confocal fluorescence microscopy showing reduced numbers of cholinergic neurons in the DMV of ChAT Cre mice receiving an injection of AAVrg-FLEX-dtA compared with the controls. Scale bar, 80 μm, cc: central canal, 12N: hypoglossal nucleus. (C) Summary graph showing the number of cholinergic neurons on the left and right sides of the DMV in the control (n = 4 mice) and experimental mice (n = 4 mice). Unpaired t test, **p < 0.01. (D) Images showing a lack of vAChT-positive nerve terminals in the liver parenchyma of ChAT Cre + AAVrg-FLEX-dtA mice (right panel). Left panel: control. Scale bar, 50 μm. (E, F) Summary plots showing a significant decrease in hepatic (control, n = 7 mice; experimental mice, n = 6 mice), but not plasma (control, n = 11 mice; experimental mice, n = 8 mice), ACh contents in ChAT Cre + AAVrg-FLEX-dtA mice compared to the controls. Unpaired t test, *p < 0.05. The data supporting the graphs shown in the figure (Fig 2C, 2E, and 2F) are available in the S1 Data file. AAV, adeno-associated virus; vAChT, vesicular acetylcholine transporter.

The role of DMV cholinergic neurons innervating the liver in controlling hepatic lipid metabolism remains unexplored. Consequently, we examined the physiological impact of hepatic cholinergic loss-of-function on hepatic fat accumulation. First, to selectively target liver-projecting cholinergic neurons, we used a retrograde AAV (AAVrg) encoding a Cre-dependent diphtheria toxin subunit A fragment (dtA) that induces apoptosis of target cells [ 23 ]. AAVrg-FLEX-dtA and AAVrg-FLEX-tdTomato as control were injected into the livers of the ChAT Cre mice ( Fig 2A ). To validate cholinergic cell death in the DMV, we sacrificed mice at 10 weeks post-viral inoculation and stained the DMV cholinergic neurons with an anti-ChAT antibody. The number of DMV cholinergic neurons was significantly lower in ChAT Cre mice that received retroAAV-FLEX-dtA than in the controls (Figs 2B, 2C , S2A , and S2B ). This reduced number of DMV cholinergic neurons was strongly associated with a lack of vAChT-positive nerve terminals in the livers of ChAT Cre mice that received AAVrg-FLEX-dtA ( Fig 2D ). Importantly, measurement of ACh levels revealed a significant reduction in hepatic ACh content in the experimental mice relative to controls, whereas there was no difference in plasma ACh levels between the experimental and control groups ( Fig 2E and 2F ). These results support the interpretation that our experimental approach effectively ablates parasympathetic cholinergic neurons innervating the liver.

In addition to cholinergic input, RT-qPCR of liver tissue homogenates revealed the presence of muscarinic ACh receptor types 1, 2, 3, 4, and 5 (Chrm1, 2, 3, 4, and 5) ( S1A Fig ) consistent with our prior study [ 16 ]. Western blot analysis of liver tissue homogenates showed the expression of CHRM1, 2, 4, and 5, but not CHRM3 ( S1B and S1C Fig ). As CHRM4 protein expression was highly expressed compared to other CHRM subtypes, we immunolabeled the liver tissues with an anti-CHRM4 antibody. CHRM4 was detected in areas adjacent to vAChT-positive terminals ( S1D Fig ). Similarly, CHRM2 was also found in hepatocytes ( S1E Fig ). Taken together, our analyses of cleared liver tissues using 3D imaging techniques, as well as antero- and retrograde neural tracing robustly support the interpretation that the liver is innervated by parasympathetic cholinergic neurons.

Given that DMV cholinergic neurons projected to the liver, we injected an anterograde AAV1 encoding Cre-dependent tdTomato and a synaptophysin-GFP (sypGFP) fusion protein separated by the sequence of a T2A self-cleaving peptide (AAV1-FLEX-tdTomato-T2A-sypGFP [ 22 ]) into the DMV of ChAT Cre mice ( Fig 1C ). Under these experimental conditions, we anticipated that tdTomato would be expressed in DMV cholinergic neurons and that sypGFP would move forward to the axon terminals of DMV cholinergic neurons. As shown in Fig 1E , immunostaining revealed that the DMV cholinergic neurons were positive for tdTomato, indicating that the DMV cholinergic neurons were successfully infected. Immunolabeling of liver sections further revealed numerous sypGFP-positive nerve terminals in a subset of hepatocytes and bile duct epithelial cells cholangiocytes ( Fig 1E and 1F ). SRY-Related HMG Box Gene 9 (SOX9)-positive cholangiocytes were surrounded by vAChT-positive nerve terminals ( Fig 1F ). 3D imaging analysis of cleared liver tissue collected from ChAT Cre mice injected with AAV1-FLEX-tdTomato-T2A-sypGFP further confirmed the presence of cholinergic nerve terminals around the portal vein and in a subset of hepatocytes ( Fig 1G and 1H and S2 Movie ), consistent with a recent findings of parasympathetic cholinergic innervation of the bile duct in human liver tissues [ 15 ].

(A) Schematic illustration of our experimental conditions (upper panel). Bottom panel: Images of confocal fluorescence microscopy showing co-expression of tdTomato and ChAT in a subset of DMV cholinergic neurons in ChAT Cre mice receiving an injection of AAV.Php.eB-tdTomato into the liver, representing cholinergic neurons innervating the liver (white arrowheads). Scale bar, 30 μm. (B) Schematic illustration of our experimental conditions (upper panel). Bottom panel: Images showing PLAP-positive nerve fibers in the liver parenchyma. Right panel: Higher-magnification view of the black square box in the left panel. Scale bars, 100 μm (left), 20 μm (right). (C) Images of confocal fluorescence microscopy showing the expression of vAChT-positive nerve terminals (green) in the liver parenchyma (inset: higher-magnification view of vAChT-positive terminals). Scale bar, 70 μm. (D) Schematic illustration of our experimental conditions. (E) Images of confocal fluorescence microscopy showing tdTomato-positive cholinergic neurons in the DMV (upper panel; white arrowheads) and sypGFP-positive nerve terminals (bottom panel; white arrowheads) in the liver of ChAT Cre mice injected with AAV1-FLEX-tdTomato-T2A-sypGFP into the DMV. A small number of hepatocytes and cholangiocytes received parasympathetic cholinergic innervation (inset). Blue: nucleus staining with DAPI. Upper panel: scale bar, 30 μm; lower panel: left, scale bar, 20 μm; right, 40 μm. (F) Images of confocal fluorescence microscopy showing that SOX9-positive cholangiocyte cells receive vAChT-positive nerve terminals (white arrowheads). Scale bar: 10 μm. (G) Image of the liver after tissue clearing. (H) Z-stack image showing parasympathetic GFP-positive cholinergic nerve terminals (green) around the periportal area in cleared liver tissue collected from ChAT Cre mice injected with AAV1-FLEX-tdTomato-T2A-sypGFP (upper panel). Bottom panel: 2D image of GFP-positive cholinergic nerve terminals shown in the upper panel. White arrowheads indicate the cholinergic nerve terminals. Scale bar: 50 μm. AAV, adeno-associated virus; PLAP, placental alkaline phosphatase; vAChT, vesicular acetylcholine transporter.

We subsequently examined whether these DMV cholinergic neurons send axonal projections to the liver by employing a retrograde AAV that encodes a Cre-dependent placental alkaline phosphatase (PLAP), which is expressed on the plasma membrane (AAVrg-FLEX-PLAP). Injection of this AAVrg-FLEX-PLAP into the liver of ChAT Cre mice resulted in the presence of PLAP-positive nerves in the parenchyma of the liver, particularly in the periportal and midlobular areas ( Fig 1B ). Immunostaining with an antibody against the presynaptic cholinergic marker vesicular acetylcholine transporter (vAChT) revealed the presence of vAChT-positive nerve terminals scattered throughout the liver parenchyma, but higher levels of vAChT-positive terminals were observed around the periportal zones ( Fig 1C ). In addition, the 3D projection view of the cleared liver tissue from the ChAT Cre ;channelrodopsin (ChR2)-tdTomato mice showed the presence of hepatocytes receiving tdTomato-positive puncta-like nerve terminals ( S1 Movie ). Consequently, our findings align with prior studies indicating that a subset of DMV cholinergic neurons innervate the liver [ 16 , 20 , 21 ].

In our previous study [ 16 ], we showed that a subset of DMV cholinergic neurons projected to the liver. To extend and further confirm the presence of DMV cholinergic neurons innervating the liver, we injected a Cre-dependent viral tracer (adeno-associated virus (AAV).PHP.eB-FLEX-tdTomato [ 17 ]) into the liver of choline acetyltransferase (ChAT)-IRES-Cre (ChAT Cre ) mice [ 18 ]. The ChAT Cre transgenic strain has been used to selectively label parasympathetic cholinergic neurons [ 19 ]. At 3 weeks post-viral infection, we performed double immunocytochemistry with anti-ChAT and anti-tdTomato antibodies on the brain sections and found that the DMV displayed a small number of tdTomato-positive cells ( Fig 1A ). Importantly, all tdTomato-positive cells in the DMV were ChAT-expressing cells (n = 72 out of 72 neurons from 3 mice), and approximately 10% of DMV cholinergic neurons expressed tdTomato (range, 7% to 14%; n = 72 out of 660 cholinergic neurons from 3 mice).

Discussion

The current study provides concrete evidence for the existence of the cholinergic system in the mouse liver. Our study showed that DMV cholinergic neurons directly projected to hepatocytes and cholangiocytes and that hepatocytes expressed muscarinic ACh receptors. Our 3D imaging analysis of cleared liver tissue also revealed the presence of cholinergic nerve terminals around the portal vein. Additionally, our study found that deleting cholinergic neurons innervating the liver prevented the development of hepatic steatosis during HFD feeding, which was closely linked to the browning of ingWAT. ChATCre mice without cholinergic input to the liver displayed elevated bile acid synthesis, primarily through the CYP7B1-mediated alternative pathway, leading to elevated serum bile acid levels. Importantly, the experimental mice exhibited elevated Tgr5 gene expression in ingWAT, which was found to be critical in mediating the beneficial effects of the loss of hepatic cholinergic function. Specifically, knockdown of the Tgr5 gene in ingWAT completely abolished these beneficial effects and led to the reappearance of hepatic steatosis in the experimental groups. These findings suggest that TGR5 is a crucial mediator of the metabolic benefits conferred by loss of hepatic cholinergic function. Taken together, our results suggest that the brain–liver axis plays an essential role in BA synthesis and secretion. The interorgan crosstalk via bile acids is also critical for the development of hepatic steatosis.

The liver plays a major role in controlling energy metabolism, including macronutrient metabolism, lipid homeostasis, and energy storage [10]. Like other visceral organs [53–55], the liver is innervated by the vagus nerve [56], and vagal innervation to the liver regulates these metabolic functions [5,8,57,58]. Parasympathetic (or vagal) efferent neurons innervating the liver are located in the DMV [59]. Prior anatomical studies with retrograde neuronal tracers such as cholera toxin B, pseudorabies virus, and AAV encoding a Cre-dependent reporter protein demonstrated that the mouse liver receives parasympathetic nerve innervation from the DMV [16,20,21]. DMV motor neurons are acetylcholine (ACh)-expressing neurons [53]. We recently showed that hepatocytes received direct DMV cholinergic input and expressed muscarinic ACh receptor genes [16]. Importantly, a short-term change in the activity of DMV cholinergic neurons projecting to the liver regulated hepatic glucose output in lean mice [16]. In contrast to these prior findings, recent 3D imaging studies revealed a paucity of parasympathetic cholinergic innervation in the mouse liver [4,60], indicating that hepatic metabolism is primarily regulated by the sympathetic nervous system. However, a recent elegant study by the Tang research group clearly demonstrated the presence of vAChT-positive nerve terminals in the bile duct of human liver tissues, suggesting that the liver receive parasympathetic cholinergic innervation [15].

In fact, accumulated studies have shown that parasympathetic innervation to the liver controls hepatic glucose output, lipid metabolism, and bile formation [5,7,8,57,61]. However, there is limited information available on the detailed neuroanatomy of parasympathetic innervation of the liver, leading some to question its importance in controlling hepatic metabolism [4,60,62]. In addition, although hepatic branch vagotomy altered multiple physiological processes, including glucose homeostasis [57,61], hepatic lipid metabolism [5,7], and hepatic inflammatory response [8], studies involving hepatic branch vagotomy have not provided conclusive evidence supporting the role of the parasympathetic cholinergic system in liver function. The hepatic branches of the vagus nerve also contained sensory nerves that transmit interoceptive information from the liver to the brain. Despite this, several studies have used virus-mediated tracing methods to clearly demonstrate the presence of DMV cholinergic neurons that innervate the liver [16,20,21].

Our study, which utilized anterograde tracing techniques alongside 2D immunostaining and 3D imaging of cleared liver tissue, uncovered direct synaptic connections between cholinergic neurons in the DMV and the liver. The elimination of DMV cholinergic neurons that innervate the liver led to a substantial reduction in the number of vAChT-positive nerve terminals and hepatic ACh levels. It is possible that differences in the morphology of preganglionic and postganglionic neurons may contribute to the discrepancy between our findings and recent 3D imaging studies [4,60]. While sympathetic postganglionic neurons and sensory neurons, particularly c-fibers, have unmyelinated nerve fibers, the nerve fibers of parasympathetic cholinergic preganglionic neurons are myelinated [63,64]. In addition, it is important to emphasize that the cholinergic nerves form a complex plexus with multiple axonal varicosities (en-passant synapses) in both the central and peripheral nervous systems [65–68]. Terminal synapses are typically established at the end of an axon’s projections, while en passant synapses are situated along the axon shaft, often at a considerable distance from the terminal. All these prior studies using the vAChT antibody showed that staining with the vAChT antibody consistently revealed puncta-like varicosities. To circumvent this issue, we expressed PLAP exclusively in liver-innervating cholinergic neurons. PLAP staining revealed the extensive cholinergic nerve fibers in the periportal and midlobular areas, further supporting the presence of hepatic cholinergic innervation.

Compared to the experimental groups, ChATCre mice fed HFD showed elevated basal and fasting glucose levels and higher basal insulin levels, which led to hyperglycemic and hyperinsulinemic conditions during HFD feeding. These conditions caused hepatic steatosis in control mice. Interestingly, the deletion of parasympathetic cholinergic input to the liver reversed the detrimental effects of high-fat feeding on insulin sensitivity and hepatic fat accumulation.

The effects observed were partly due to the decrease in hepatic de novo lipogenesis and increase in fatty acid oxidation in the livers of ChATCre mice, which lack DMV cholinergic innervation. In other words, when DMV cholinergic innervation to the liver was deleted, genes associated with lipogenesis were expressed at lower levels, while genes involved in fatty acid oxidation were expressed at higher levels, leading to reduced levels of triglycerides. Hence, our results suggest a significant role for parasympathetic cholinergic innervation in the regulation of hepatic lipid metabolism under obesogenic conditions.

Simple hepatic steatosis resulted from an imbalance between the delivery and production of fat in the liver and its subsequent secretion and metabolism [69]. Excessive dietary fat led to increased plasma concentrations of TGs and FFAs, resulting in increased uptake into hepatocytes [70]. In our preparations, both groups consumed the same amount of food, implying that there may not be a substantial difference in the daily intake of fat. Additional sources of fatty acids can also be obtained from the breakdown of TGs stored in adipocytes, which is mainly controlled by insulin [71]. Insulin regulates systemic lipid metabolism by suppressing lipolysis in adipocytes. This mechanism is the primary way that insulin controls the breakdown of fats in these cells. As a result, insulin typically increases the uptake of FFAs from lipoproteins and decreases their release from adipocytes [70]. In contrast, insulin resistance depressed both of these functions [70]. ChATCre mice, which lack DMV cholinergic neurons innervating the liver, displayed enhanced insulin sensitivity compared with the control group. This metabolic profile, including decreased adiposity, lower levels of plasma glycerol, and reduced fat mass, may contribute to the improved insulin sensitivity observed in experimental mice. By improving insulin resistance in adipocytes, the flow of FFAs and glycerol to the liver was limited, preventing the development of hepatic steatosis in ChATCre mice that lack cholinergic innervation to the liver during HFD feeding.

We should highlight that disrupting the brain–liver axis caused the browning of ingWAT, which was a process that enhances glucose uptake and significantly contributes to systemic glucose disposal. This improvement in glucose disposal can ultimately lead to better insulin sensitivity [36]. Hepatocytes secrete various signaling molecules, such as bile acids, which can trigger WAT browning through TGR5 [12,40,72]. Daily administration of the TGR5 agonist for 1 week resulted in a significant decrease in subcutaneous white adipose tissue (scWAT) mass and the up-regulation of browning marker genes, including Ucp1 and Cidea [12]. ChATCre mice lacking liver-projecting cholinergic neurons showed a significant reduction in WAT mass and adipocyte size. Interestingly, these mice also exhibited an increase in thermogenic gene and protein expression in ingWAT, leading to an elevation in nocturnal ingWAT temperature. The up-regulation of Cyp7b1 and BA levels due to the loss of hepatic cholinergic innervation may lead to browning of ingWAT as a result of crosstalk between the liver and ingWAT.

Communication between ingWAT and other organs, including the liver and muscle, is essential for the proper functioning of systemic metabolism [71]. Our current study revealed that the crosstalk between the liver and ingWAT through bile acids played a significant role in the progression of hepatic steatosis. Remarkably, silencing Tgr5 in the ingWAT completely reversed the beneficial effects of the loss of hepatic cholinergic innervation. Tgr5 knockdown resulted in enlarged adipocytes, leading to hypertrophy. Surprisingly, knockdown of Tgr5 that was restricted to ingWAT induced lipid accumulation in the livers of ChATCre mice lacking cholinergic innervation. In other words, the absence of hepatic cholinergic innervation failed to prevent the onset of hepatic steatosis following the silencing of Tgr5 in ingWAT, suggesting that the ingWAT makes a crucial contribution to the development of hepatic steatosis. In fact, knockdown of Tgr5 in ingWAT elevated plasma glycerol levels, indicating a role of TGR5 in regulating lipolysis in ingWAT. While our study provides evidence for crosstalk between the liver and ingWAT via TGR5, it is possible that TGR expressed in central neurons may also play a role in the observed effects as activation of hypothalamic TGR5 suppressed food intake and prevented DIO [73,74].

In conclusion, our study provides substantial evidence for the presence of parasympathetic cholinergic input to the liver, which constitutes a key component of the brain–liver axis. This axis is involved in the synthesis and secretion of bile acids, and the interorgan communication between the liver and ingWAT via bile acids plays a crucial role in the development of systemic insulin resistance and hepatic steatosis under obesogenic conditions. Therefore, targeting parasympathetic cholinergic input to the liver may offer a promising avenue for treating systemic insulin resistance and its associated hepatic steatosis in obesity.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002865

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