Rspo3-mediated metabolic liver zonation regulates systemic glucose metabolism and body mass in mice

Kenji Uno; Takuya Uchino; Takashi Suzuki; Yohei Sayama; Naoki Edo; Kiyoko Uno-Eder; Koji Morita; Toshio Ishikawa; Miho Koizumi; Hiroaki Honda; Hideki Katagiri; Kazuhisa Tsukamoto

doi:10.1371/journal.pbio.3002955

. 2025 Jan 24;23(1):e3002955. doi: 10.1371/journal.pbio.3002955

Rspo3-mediated metabolic liver zonation regulates systemic glucose metabolism and body mass in mice

Kenji Uno ^1,^*, Takuya Uchino ¹, Takashi Suzuki ¹, Yohei Sayama ¹, Naoki Edo ², Kiyoko Uno-Eder ², Koji Morita ¹, Toshio Ishikawa ¹, Miho Koizumi ³, Hiroaki Honda ³, Hideki Katagiri ⁴, Kazuhisa Tsukamoto ¹

Editor: Jason W Locasale⁵

PMCID: PMC11759367 PMID: 39854351

Abstract

The unique architecture of the liver consists of hepatic lobules, dividing the hepatic features of metabolism into 2 distinct zones, namely the pericentral and periportal zones, the spatial characteristics of which are broadly defined as metabolic zonation. R-spondin3 (Rspo3), a bioactive protein promoting the Wnt signaling pathway, regulates metabolic features especially around hepatic central veins. However, the functional impact of hepatic metabolic zonation, regulated by the Rspo3/Wnt signaling pathway, on whole-body metabolism homeostasis remains poorly understood. In this study, we analyze the local functions of Rspo3 in the liver and the remote actions of hepatic Rspo3 on other organs of the body by using murine models. Rspo3 expression analysis shows that Rspo3 expression patterns are spatiotemporally controlled in the murine liver such that it locates in the pericentral zones and converges after feeding, and the dynamics of these processes are disturbed in obesity. We find that viral-mediated induction of Rspo3 in hepatic tissue of obesity improves insulin resistance and prevents body weight gain by restoring attenuated organ insulin sensitivities, reducing adipose tissue enlargement and reversing overstimulated adaptive thermogenesis. Denervation of the hepatic vagus suppresses these remote effects, derived from hepatic Rspo3 induction, toward adipose tissues and skeletal muscle, suggesting that signals are transduced via the neuronal communication consisting of afferent vagal and efferent sympathetic nerves. Furthermore, the non-neuronal inter-organ communication up-regulating muscle lipid utilization is partially responsible for the ameliorations of both fatty liver development and reduced skeletal muscle quality in obesity. In contrast, hepatic Rspo3 suppression through Cre-LoxP-mediated recombination system exacerbates diabetes due to glucose intolerance and insulin resistance, promotes fatty liver development and decreases skeletal muscle quality, resulting in obesity. Taken together, our study results reveal that modulation of hepatic Rspo3 contributes to maintaining systemic glucose metabolism and body composition via a newly identified inter-organ communication mechanism.

Metabolism in the liver is divided between the pericentral and periportal zones, but how does this zonation affect whole body physiology? This study shows that proper liver zonation, which is regulated by Rspo3, contributes to glucose metabolism homeostasis.

Introduction

The unique architecture of the liver consists of hepatic lobules. These lobules have a distinct bloodstream construction, in which nutrient-rich venous blood from the intestines mixes with arterial blood around the portal triad, traveling through the hepatic sinusoids and collecting in central zones [1]. The lobular architecture divides hepatic features into 2 distinct metabolic zones, so-called “metabolic zonation” [2–4], which have spatially and temporally separate functions such as glucose (gluconeogenesis, glycolysis), lipid (β-oxidation, lipogenesis), and amino acid (AA) metabolism (glutamine synthesis). While mainly gluconeogenesis and β-oxidation are increased in the periportal zone, glycolysis, lipogenesis, and glutamine synthesis are increased in the pericentral zone [5–7]. Thus, metabolic zonation plays an important role in maintaining both intra-hepatic and systemic metabolism [8]. However, the precise regulatory mechanism underlying metabolic zonation in the liver and the pathophysiological significance of disturbances associated with nutrient overloading and their contributions to metabolic disorders, i.e., metabolic syndrome, have yet to be clarified.

Members of the R-spondin family (R-spondin1-4 (Rspo1-4)), proteins secreted by several organs, function as amplifiers of Wnt pathway activity by engaging LGR4-6 G protein-coupled receptors to increase the cell membrane density of Wnt receptors [6,9]. Wnts, also recognized as a family of secreted proteins, locally bind to their receptors, leading to nuclear accumulation of the transcription activator β-catenin and increases in downstream target gene expressions. This canonical pathway is regarded as an evolutionarily conserved cascade, playing important roles in stem cell biology and adult tissue homeostasis [10–12].

Furthermore, R-spondin3 (Rspo3) was demonstrated to be a key molecule determining metabolic zonation in the liver by regulating AA-related gene expressions such as glutamine synthase around central zones [13]. In addition to Rspo3 findings, Rspo1 was also identified as controlling metabolic zonation and hepatic size and growth via the RSPO-LGR4/5-ZNRF3/RNF43 module [14]. Overexpression or ablation of hepatic β-catenin, a downstream target of the Rspo family, in murine models on high-fat diet reportedly causes diet-induced obesity with progressive hepatic steatosis and insulin resistance or protects against diet-induced obesity, respectively [15]. In addition to these roles of the Rspo/Wnt/β-catenin signaling pathway in the liver, RSPO3 is associated with an increased body mass index-adjusted waist-to-hip ratio, impacting upper body fat distribution by stimulating abdominal adipose proliferation [16]. Ablation of LGR4, an Rspo family receptor, promoted energy expenditure and induced brown-like adipocytes in white adipose tissue (WAT) depots, i.e., white-to-brown transition [17]. These prior reports suggested that focusing on modulation of metabolic zonation in the liver via Rspo family proteins raises the possibility of elucidating inter-organ communication throughout the body.

Herein, we demonstrated that modulation of hepatic Rspo3 improves obesity-associated features, including diabetes, insulin resistance, and fatty liver. Most notably, hepatic Rspo3 reverses enlargement of WAT in obesity via a newly identified neuronal inter-organ linkage from the liver to adipose tissue. In contrast, hepatic Rspo3 suppression exacerbates obesity-associated features, including glucose intolerance, insulin resistance, and fatty liver change. Therefore, modulating and restoring metabolic zonation by targeting the hepatic Rspo3 signaling pathway may lead to novel therapeutic strategies for diabetes and metabolic syndrome.

Results

Metabolic functions of Rspo3 in the liver are disturbed in obesity

First, we examined whether serum Rspo3 levels were changed in either lean or genetically obese (ob/ob) murine models and found that Rspo3 levels did not differ significantly between these 2 conditions in fasted states (Fig 1A). In lean models, but not in obesity, serum Rspo3 levels were up-regulated by feeding (Fig 1A). Second, we examined gene expression levels of Rspo3 in the liver (Fig 1B), adipose tissues including WAT and brown adipose tissue (BAT), and skeletal muscles of both lean and obese models (Fig 1C). We found that endogenous Rspo3 expressions in the liver and WAT, the major organs producing Rspo3, were decreased, while those in BAT and skeletal muscle were not, according to the degree of obesity. Feeding led to up-regulation of hepatic Rspo3 expressions in the livers of lean model mice, while no such phenomenon was observed in obese mice (Fig 1D). These results indicated that Rspo3 levels were dynamically altered according to both physiological conditions, including lean and obese states, and nutritional settings, such as fed and fasted states.

Fig 1 — **(A)** Serum Rspo3 levels under either fasted or fed conditions were examined in lean and ob/ob mice (n = 7–14). **(B)** Hepatic Rspo3 mRNA expression (n = 4–5) and immunoblotting results of liver extracts with anti-Rspo3 and anti-GAPDH antibodies were measured in lean and ob/ob mice. The relative intensities (the ratios of Rspo3/GAPDH protein volume) were calculated. **(C)** Rspo3 mRNA expressions in several organs were measured and compared among lean, DIO, and ob/ob mice (n = 4–8). **(D)** Hepatic Rspo3 mRNA expressions under either fasted or fed conditions were measured in lean and ob/ob mice (n = 5–13). **(E)** Representative immunofluorescence analysis images of the pericentral zone in the liver with anti-Rspo3, anti-HNF4α, anti-CD31, anti-CK19 antibodies, and DAPI staining. The scale bars indicate 100 μm. **(F)** Representative immunofluorescence analysis images of the pericentral zone in the liver with anti-Rspo3 antibody and DAPI staining. The scale bars indicate 50 μm. **(G)** The intensity of Rspo3 immunofluorescence was measured on 4 lines, each 20 μm from the hepatic central veins, obtained randomly (hepatic central veins; n = 29–30, 3 mice per group). **(H)** Representative RNA-Scope analysis images of the pericentral zone in the liver with specific Rspo3 probe. The scale bars indicate 100 μm. **(I)** The numbers of Rspo3-positive signals (dots) were measured randomly (hepatic central veins; n = 13–21, 3–4 mice per group). The data underlying the graphs shown in the figure can be found in **S1 Data**. Data are presented as means ± SD. *P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01, ††P < 0.01 by the unpaired t test. CD31, cluster of differentiation 31; CK19, Cytokeratin 19; DIO, diet-induced obese; HNF4a, Hepatocyte nuclear factor 4a; Rspo3, R-spondin3.

Next, we examined the localization of Rspo3 expression in hepatic lobules. To distinguish between the zones around central veins (pericentral) versus portal veins (periportal), we subjected liver sections to Azan staining, allowing the periportal zone to be detected (S1 Fig). Analysis of immunofluorescence (IF) of liver sections revealed much more Rspo3 expression in pericentral hepatocytes than in periportal hepatocytes (S1 Fig), suggesting the existence of zonation regulating hepatic Rspo3 expression and localization. Comparison of IF between Rspo3 and hepatocyte nuclear factor 4α (HNF4α, hepatocyte marker) confirmed that HNF4α-positive hepatocytes simultaneously possess Rspo3 IF reactivity (Fig 1E). Comparisons of IF between Rspo3 and cluster of differentiation 31 (CD31, endothelial cell marker) confirmed Rspo3-positive cells to be present at much lower densities in central vein endothelial cells than in hepatocytes (Fig 1E). Comparisons of IF between Rspo3 and cytokeratin 19 (CK19, sinusoidal endothelial marker) confirmed that there were no Rspo3-positive cells among neighboring sinusoidal endothelial cells (Fig 1E). Furthermore, Rspo3-positive hepatocytes in lean models were more responsive to stimulation or were increased in number, especially in the pericentral zone, in the fed than in the fasted state, and that the cellular dynamics of feeding on cumulative Rspo3 localization in the pericentral zone were disturbed in obese models (Figs 1F and S2). The localization of Rspo3-positive hepatocytes in obesity was rather widespread, extending from the pericentral zone to the periportal zone (Figs 1F and S2). In addition, the intensity of Rspo3 IF in hepatocytes in the pericentral zones, reflecting the extent of Rspo3 expression, indicated that feeding increased the intensity of Rspo3 IF, especially in lean models, while minimally impacting obese models (Fig 1G). Furthermore, to investigate the expression patterns of Rspo3 mRNA in the liver under these conditions such as lean versus obese states, or fasted versus fed states, we performed RNA-Scope, a newly developed technique of in situ hybridization (ISH), by using the specific probe against Rspo3. RNA-Scope revealed far more Rspo3-positive signals in the pericentral zones than in the periportal zones (Fig 1H), observations consistent with the results of IF (Fig 1F). In the pericentral zones, Rspo3-positive signals existed in endothelial cells as well as hepatocytes, reflecting the difference in Rspo3 expression patterns between mRNA and protein levels. On the other hand, the numbers of Rspo3-positive signals were increased in the pericentral zones of lean models after feeding, showing disturbance of the dynamics of feeding to change Rspo3 mRNA localization toward these zones, especially in obese models (Fig 1I).

Thus, the convergence of Rspo3-positive hepatocytes, at either the mRNA or the protein level, toward the pericentral zone in response to feeding (i.e., spatiotemporal characteristics of Rspo3) appears to play an important role in regulating intra-hepatic metabolism under normal conditions, a phenomenon which is dysregulated in pathophysiological states such as obesity.

Rspo3 influences systemic glucose metabolism and body composition

To investigate whether hepatic Rspo3 functions in regulating metabolic physiology in mammals, we overexpressed Rspo3 in the livers of C57BL/6 mice fed normal chow diets (Rspo3-mice) using adenoviral gene transduction. Systemic infusion of Rspo3 recombinant adenovirus resulted in selective transgene expression in the liver (1.46-fold of protein levels as compared to control mice) (Fig 2A), while there were no differences in Rspo3 expressions in other organs (S3A Fig). Mice given the lacZ adenovirus were used as controls (LacZ-mice). Hepatic Rspo3 induction significantly increased gene expressions of LGR5 and Axin2, downstream targets of the Rspo3/Wnt signaling pathway (S3B Fig). While hepatic Rspo3 induction increased gene expressions of ornithine aminotransferase (Oat), cholesterol 7alpha-hydroxylase (Cyp7a1), and retinol dehydrogenase9 (Rdh9) [13], which are expressed and function mainly in the pericentral zones (S3C Fig), it decreased hydroxysteroid 17-beta dehydrogenase (Hsd17) and cytochrome P450 2f2 (Cyp2f2) [13], which are expressed and function mainly in the periportal zones (S3D Fig). These results indicated hepatic Rspo3 induction to up-regulate the Rspo3/Wnt signaling pathway in the liver and thereby shifted metabolic gene expressions, a process by which hepatic features of metabolism are considered to move from the periportal to the pericentral zone. Under these conditions, hepatic Rspo3 expression significantly decreased fasting blood glucose and insulin levels (Fig 2B), producing both a decrease in the expression of a major hepatic gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK) and an increase in the expression of the hepatic glycolytic enzyme glucokinase (GK) (Fig 2C). While hepatic expressions of lipogenic genes such as sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthetase (FASN), and peroxisome proliferator-activated receptor (PPAR) γ, were increased, hepatic expressions of lipogenic genes such as stearoyl CoA desaturase 1 (SCD1) were decreased in Rspo3-mice (Fig 2D). Hepatic expressions of lipogenic and lipid utilizing genes, such as PPARδ, and lipid oxidative genes, such as carnitine palmitoyltransferase 1a (Cpt1a), but not PPARα, were also increased in Rspo3-mice (Fig 2D). Under these conditions, hepatic Rspo3 expression altered liver weight (Fig 2E) with no change in liver histology, as demonstrated by hematoxylin eosin (HE) staining (Fig 2F). In agreement with previous reports showing Rspo3 to have cell proliferative capacity [14,18], hepatic Rspo3 induction increased Ki67 mRNA levels 10- or 5-fold compared to those of control mice on day 7 or 23 after adenovirus administration, respectively (S3E Fig). These results suggested that hepatic Rspo3 induction dramatically altered the types of gene expressions in the liver (shown in Fig 2C and 2D), some of which were still present on day 23 after adenovirus administration (S3F Fig), giving rise to the feature of localization in the periportal zone toward a more pericentral zone. Glucose and insulin tolerance tests revealed that Rspo3-mice exhibited increased systemic glucose tolerance and insulin sensitivity, respectively (Fig 2G and 2H), the beneficial effects of which persist for 20 days after adenovirus administration (S3G and S3H Fig). To explore whether hepatic Rspo3 induction influences whole-body energy expenditure, we examined oxygen consumption (VO₂) and the respiratory quotient (RQ), finding no VO₂ difference while RQ was decreased in Rspo3-mice (Fig 2I). Furthermore, hepatic Rspo3 induction significantly decreased the weight of epididymal WAT (Fig 2J) due to increased β3-adrenergic receptor (β3AR), UCP1 and PGC1α expressions in epididymal WAT (Fig 2K) and/or decreased food intake (Fig 2L), contributing to the prevention of body weight gain for 7 days after adenoviral treatment (Fig 2M). Collectively, these results suggest hepatic Rspo3 to play an important role in the regulation of systemic glucose metabolism and body composition.

Fig 2 — **(A–M)** Rspo3 (red bars and circles) or LacZ (blue bars and circles) adenovirus was administered to standard diet-fed control mice. **(A)** Hepatic Rspo3 mRNA expression (n = 5) and immunoblotting of liver extracts with anti-Rspo3 and anti-GAPDH antibodies and the relative intensities (the ratios of Rspo3/GAPDH protein volume), **(B)** fasting blood glucose (n = 4–5) and insulin levels (n = 5–9), **(C)** hepatic PEPCK and GK mRNA expressions (n = 5–7), **(D)** hepatic SREBP1c, FASN, PPARγ, SCD1, PPARα, PPARδ, and Cpt1a mRNA expressions (n = 7), **(E)** liver weight (n = 6–7), and **(F)** representative histological analysis images of the liver (HE staining) were examined on day 7 after adenovirus administration. The scale bars indicate 100 μm. **(G)** Glucose tolerance (n = 5–11) and **(H)** insulin tolerance (n = 6) tests were performed on day 7 after adenovirus administration. **(I)** VO₂ and RQ (n = 5–6) were measured on day 4 after adenovirus administration. **(J)** Epididymal WAT weight (n = 7), **(K)** β3AR, UCP1, and PGC1α mRNA expressions in epididymal WAT (n = 5–7); **(L)** food intake (n = 5); and **(M)** body weight gain (n = 11–12) were examined on day 7 after adenovirus administration. Body weight changes from day 0 to day 20 after adenovirus administration were measured (n = 5). The data underlying the graphs shown in the figure can be found in **S1 Data**. Data are presented as means ± SD. *P < 0.05, **P < 0.01 by the unpaired t test. b3AR, b3-adrenergic receptor; Cpt1a, carnitine palmitoyltransferase 1a; FASN, fatty acid synthetase; FBG, fasting blood glucose; GK, glucokinase; HE, hematoxylin eosin; PPAR, peroxisome proliferator-activated receptor; PEPCK, phosphoenolpyruvate carboxykinase; RQ, respiratory quotient; Rspo3, R-spondin3; SCD1, stearoyl CoA desaturase 1; SREBP1c, sterol regulatory element-binding protein 1c; WAT, white adipose tissue.

Rspo3 improves obesity-induced metabolic disorders locally in the liver

Next, to investigate whether hepatic Rspo3 induction has any effects on obesity-related metabolic dysregulation, we administered Rspo3 adenovirus to obese (ob/ob) mice (obese Rspo3-mice). Systemic infusion of Rspo3 adenovirus resulted in selective transgene expression in the liver (1.85-fold of protein levels as compared to obese control mice), to the same or a degree slightly lower than that of endogenous Repo3 expression in lean control mice (0.74-fold) (Fig 3A). There were no differences in Rspo3 expressions in other organs (S4A Fig). RNA-Scope revealed that Rspo3-positive signals are also increased in obese Rspo3-mice (3.6-fold) (S4B Fig) to a lesser degree than indicated by the quantitative RT-PCR results (Fig 3A). However, hepatic Rspo3 induction did not alter circulating Rspo3 levels on day 7, showing only a temporary increase on day3, after adenovirus administration as compared to the level in control mice (S4C Fig). As in lean Rspo3-mice, hepatic Rspo3 induction significantly increased gene expressions of LGR5 and Axin2, downstream targets of the Rspo3/Wnt signaling pathway (S4D Fig), as well as gene expressions of Oat and Rdh9, markers of genes in the pericentral zone (S4E Fig), while it decreased Cyp2f2, a marker of genes in the periportal zone (S4F Fig). According to the RNA-sequence results, other markers of genes in the pericentral zone, such as Cyp2a4 (1.76-fold, P < 0.01), Cyp1a2 (2.00-fold, P < 0.01), Rnase4 (3.87-fold, P < 0.01), Cyp7a1 (1.19-fold, P < 0.05), Gstm3 (4.07-fold, P < 0.01), and Avpr1 (1.59-fold, P < 0.05), were increased, while other markers of genes in the periportal zone, such as Arg1 (0.75-fold, P < 0.01), Gls2 (0.20-fold, P < 0.01), Aldh1b1 (0.81-fold, P < 0.01), and Sds (0.28-fold, P < 0.01), were decreased in obese Rspo3 mice (S1 Table). All of these results suggested the reprograming of hepatocytes from periportal to pericentral features.

Fig 3 — **(A–N)** Rspo3 (red bars) or LacZ (blue bars) adenovirus was administered to standard diet-fed ob/ob mice. **(A)** Hepatic Rspo3 mRNA expression (n = 6) and immunoblotting of liver extracts with anti-Rspo3 and anti-GAPDH antibodies and the relative intensities (the ratios of Rspo3/GAPDH protein volume), **(B)** fasting blood glucose and insulin levels (n = 5–7), and **(C)** hepatic PEPCK and GK mRNA expressions were examined on day 7 after adenovirus administration (n = 6–7). **(D)** Insulin-stimulated serine473 phosphorylation of Akt protein in the liver is presented. Mice fasted 16 h were injected intravenously with insulin or vehicle alone. The liver was removed 300 s later and lysates were immunoprecipitated with the indicated antibodies, followed by SDS-PAGE and immunoblotting with anti-phospho-Akt (Ser473) antibody, or just the antibody against Akt. The relative intensities (the ratios of p-Akt/Akt protein volume) were examined. **(E)** Immunoblotting of nuclear extracts with anti-Foxo1 and anti-HDAC1 antibodies and the ratios of Foxo1/HDAC1 protein volume; and cytoplasm extracts with anti-Foxo1, anti-PDK4, and anti-GAPDH antibodies; and the relative intensities (the ratios of Foxo1/GAPDH or PDK4/GAPDH protein volume); **(F)** hepatic SREBP1c, FASN, PPARγ, and SCD1 mRNA expressions (n = 7); and **(G)** liver weight were examined on day 7 after adenovirus administration (n = 5–7). **(H)** Representative histological analysis images of the liver, HE staining, Oil red-O staining, and PAS staining on day 5 after adenovirus administration. The scale bars indicate 200 μm. **(I)** Hepatic TG contents (n = 6–7); **(J)** hepatic PPARα, PPARδ, and Cpt1a mRNA expressions (n = 7); **(K)** immunoblotting of liver extracts with anti-PPARδ, anti-Cpt1a, and anti-GAPDH antibodies and the relative intensities (the ratios of PPARδ/GAPDH or Cpt1a/GAPDH protein volume); **(L)** hepatic glycogen content (n = 7); **(M)** body weight gain (n = 5–7) and body weight changes from day 0 to day 23 after adenovirus administration (n = 4–5); and **(N)** food intake (n = 4–5) were determined on day 7 after adenovirus administration. The data underlying the graphs shown in the figure can be found in **S1 Data**. Data are presented as means ± SD. *P < 0.05, **P < 0.01 by the unpaired t test. Cpt1a, carnitine palmitoyltransferase 1a; FASN, fatty acid synthetase; FBG, fasting blood glucose; GK, glucokinase; HE, hematoxylin eosin; PAS, periodic acid chiff; PEPCK, phosphoenolpyruvate carboxykinase; PPAR, peroxisome proliferator-activated receptor; Rspo3, R-spondin3; SCD1, stearoyl CoA desaturase 1; SREBP1c, sterol regulatory element-binding protein 1c; TG, triglyceride.

Meanwhile, comparing RNA-sequence results of lean mice with those of obese mice revealed that obesity actually alters several metabolic processes including glucose and lipid metabolism, as demonstrated by gene ontology (GO) studies and KEGG pathway analyses (S5 and S6 Figs). The gene expressions of LGR5, Axin2, Oat, Cyp7a1, and Rdh9 as well as those of other marker genes in the pericentral zone, such as Cyp1a2 (0.42-fold, P < 0.01), Rnase4 (0.30-fold, P < 0.01), Rdh9 (0.68-fold, P < 0.01), and Avpr1 (0.12-fold, P < 0.01), were decreased, while the marker gene in the periportal zone, Aldh1b1 (1.65-fold, P < 0.01), was increased in obese mice as compared to lean mice (S7A–S7C Fig and S2 Table). Considering the data obtained from models of hepatic Rspo3 induction in obese mice (S4D–S4F Fig and S1 Table), these results show obesity to influence the spatially regulated expression patterns of marker genes in the pericentral and periportal zones of the liver. This pathophysiological phenomenon was suggested to be partially reversed by hepatic Rspo3 induction in these obese mice.

To investigate whether hepatic Rspo3 induction alters endogenous or exogenous Rspo3 localization in the liver on day 23 after adenovirus administration, we compared the staining patterns between Rspo3 IF, as a marker of endogenous plus exogenous Rspo3, and adenovirus-derived ZsGreen1 fluorescence, as a marker of exogenous Rspo3. Surprisingly, the results indicated much more adenovirus-derived Rspo3 to be present locally in the pericentral zones, leading to the expansion of Rspo3-positive hepatocytes, including endogenous or exogenous Rspo3, toward the periportal zones, as compared to those of control mice (S4G Fig). On the other hand, hepatic Rspo3 induction increased Ki67 IF intensity and Ki67 mRNA levels at this time point as compared to control mice (S4H and S4I Fig), observations compatible with those of previous reports describing Rspo3 as having cell proliferative capacity [14,18].

Metabolome analysis revealed hepatic Rspo3 induction as dramatically changing metabolic profiles, indicating that Rspo3 suppresses hepatic gluconeogenesis and increases glycolysis, glycogenesis, and synthesis of AAs (S8 and S9 Figs). RNA-sequence analysis also revealed that hepatic Rspo3 induction dramatically influenced several metabolic processes including glucose and lipid metabolism, as demonstrated by GO studies (S10 Fig). Under these conditions, hepatic Rspo3 induction significantly decreased fasting blood glucose and insulin levels (Fig 3B), while decreasing PEPCK expression and increasing GK expression (Fig 3C), indicating down-regulation of hepatic gluconeogenesis and up-regulation of glycolysis, respectively, results compatible with those of metabolome and RNA-sequence (PEPCK 0.66-fold, P < 0.01, and GK 1.54-fold, P < 0.01) analyses (S9 and S11 Figs). The examination of insulin signaling demonstrated hepatic Rspo3 induction to up-regulate insulin-stimulated serine473 phosphorylation of Akt in the livers of ob/ob mice (Fig 3D), which was also observed in another genetically obese (KK-Ay) mouse model (S12A Fig). Hepatic Rspo3 induction decreased Foxo1 expression in the nucleus (0.39-fold, P < 0.01) with no change of its expression in the cytoplasm of hepatocytes (Fig 3E). This restoration of hepatic insulin signaling resulted in increased Foxo1 exclusion out of the nucleus and decreased PDK4 expression in the cytoplasm of hepatocytes (0.59-fold, P < 0.01) (Fig 3E), leading to decreased hepatic gluconeogenesis [19] and increased hepatic glucose utilization in the TCA cycle [20], respectively. In support of the current research findings, previous studies demonstrated that insulin inhibits Foxo1 and allows β-catenin to bind the Wnt signaling pathway effector TCF7L2, leading to down-regulation of PEPCK expression and thereby decreasing hepatic gluconeogenesis in the postprandial state [21].

Although hepatic Rspo3 induction up-regulated the expressions of SREBP1c and FASN (Fig 3F) as well as those of FASN (1.51-fold, P < 0.01) and other lipogenic genes examined by RNA-sequence analysis (S11 Fig), without changing PPARγ expression (Fig 3F), and slightly increased liver weight (Fig 3G), histological analysis of HE and Oil red-O stained specimens revealed the size and number of lipid droplets to be decreased in Rspo3-mice (Fig 3H), observations compatible with the decreased hepatic triglyceride (TG) contents in obese Rspo3 mice (Fig 3I). Interestingly, RNA-sequence analysis revealed that hepatic expressions of PPARδ (1.31-fold, P < 0.05), which regulates the de novo lipogenic pathway in the liver and fatty acid utilization in skeletal muscle [22], and Cpt1a (1.21-fold, P < 0.05), which regulates fatty acid oxidation and mitochondrial biogenesis in the liver [23], were elevated in obese Rspo3-mice (S11 Fig). Previous studies demonstrated that Wnt/β-catenin signaling serves as an upstream regulator for PPARδ expression [24] and drives fatty acid oxidation in dendritic cells by up-regulating Cpt1a expression [25]. This observation was followed by confirmation of elevations of mRNA expressions of PPARδ and Cpt1a, but not of PPARα (Fig 3J), as well as protein expressions of PPARδ (1.99-fold, P < 0.01) and Cpt1a (2.39-fold, P < 0.05), in obese Rspo3-mice (Fig 3K), partially accounting for the amelioration of obesity-related hepatic steatosis. In addition, according to RNA-sequence analysis, hepatic expression of SCD1, a lipogenic enzyme catalyzing the synthesis of monounsaturated fatty acids such as oleate and palmitoleate, was decreased (0.57-fold, P < 0.01) (S11 Fig), and this was followed by confirmation of decreased SCD1 expression in obese Rspo3-mice (Fig 3F). As previously reported, SCD1 deficiency increased fatty acid oxidation through AMPK activation and protected against fatty liver development induced by high-fat diet feeding [26], suggesting that decreased SCD1 expressions might have contributed to the amelioration of fatty liver findings in obese Rspo3-mice. Thus, hepatic Rspo3 induction, also in obese mice, was demonstrated to dramatically alter the types of gene expressions in the liver (shown in Fig 3C, 3F, and 3J), some of which were still present on day 23 after adenovirus administration (S12B Fig).

Furthermore, periodic acid–Schiff (PAS) staining results suggested that hepatic Rspo3 expression increased glycogen storage in the liver (Fig 3H). Thus, we calculated hepatic glycogen contents, which indicated that hepatic glycogen storage was increased in obese Rspo3-mice (Fig 3L), a phenomenon consistent with the profile obtained from metabolome analysis. In detail, metabolome analysis revealed the glucose-6-phosphate (G6P) level to be increased (1.8-fold, P < 0.01) and the conversion of G6P to glucose-1-phosphate (G1P), the first step of the glycogen synthesis pathway in the liver, was also up-regulated (1.2-fold, P < 0.05) in obese Rspo3-mice, followed by an increase in UDP-glucose synthesis (1.7-fold, P < 0.01) from G1P, the next step of this pathway (S8 and S9 Figs). Based on these results, we can reasonably speculate that hepatic Rspo3 induction locally diminishes obesity-related fatty liver progression by balancing the degrees of lipogenesis, lipid oxidation, and glucose utilization (gluconeogenesis, glycolysis, and glycogenesis) in the liver (S12C Fig).

Interestingly, hepatic Rspo3 induction suppressed obesity-induced body weight gain for 7 and 23 days with decreased cumulative food intakes for 7 and 14 days after adenoviral treatment (Fig 3M and 3N). Therefore, Rspo3, locally in the liver, functions as a key molecule suppressing the progression of obesity-related fatty liver, and possesses an inherent ability to influence the systemic management of body composition, leading to maintenance of glucose metabolism at the whole-body level.

Rspo3 ameliorates the systemic glucose metabolism and body composition abnormalities in obese mice

As for glucose metabolism, hepatic Rspo3 induction decreased fasting blood glucose and serum insulin levels (Fig 3B), observations consistent with both decreased expression of PEPCK and increased expression of GK in the liver (Fig 3C). Glucose tolerance and insulin tolerance tests indicated that hepatic Rspo3 induction improved obesity-induced glucose intolerance and insulin resistance, respectively, in ob/ob mice (Fig 4A and 4B), observations also made in DIO and KK-Ay mice (S13A–S13C Fig). These improvements of glucose tolerance by hepatic Rspo3 induction were also observed in obese mice on day 20 after adenovirus administration (S13D and S13E Fig), with the beneficial effects persisting for 20 days. Specifically, the examination of insulin signaling demonstrated that hepatic Rspo3 induction up-regulated insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) or serine473 phosphorylation of Akt in remote organs, such as skeletal muscle and epididymal WAT (Fig 4C), as well as in the livers (Fig 3D), of ob/ob mice. Improvements of insulin signaling in remote organs were also more evident in KK-Ay mice (S13F Fig). These results indicated that hepatic Rspo3 induction improved obesity-induced insulin resistance throughout the body, leading to amelioration of diabetes.

Fig 4 — **(A–L)** Rspo3 (red bars and circles) or LacZ (blue bars and circles) adenovirus was administered to standard diet-fed ob/ob or KK-Ay mice. **(A)** Glucose tolerance and **(B)** insulin tolerance tests were performed on day 7 after adenovirus administration (n = 5–7). **(C)** Insulin-stimulated serine473 phosphorylation of Akt protein in skeletal muscle (hindlimb muscles) and WAT were immunoblotted with anti-phospho-Akt (Ser473) antibody, or just the antibody against Akt. Insulin-stimulated tyrosine phosphorylation of IR protein in WAT was immunoblotted with anti-phospho-Tyrosine antibody, or just the antibody against IR. The relative intensities (the ratios of p-Akt/Akt or p-Try/IR protein volume) were indicated in the p-Akt or p-Tyr panels, respectively. **(D)** Epididymal WAT weight (n = 5–7) and **(E)** β3AR, UCP1, and PGC1α expressions in epididymal WAT (β3AR; n = 6–7, UCP1; n = 12–13, PGC1α; n = 7) were examined on day 7 after adenovirus administration. **(F)** NE turnover in WAT of KK-Ay mice (n = 6–8) was performed on day 4 after adenovirus administration. **(G)** β3AR, UCP1, PGC1α, BCAT2, and BCKDH mRNA expressions in BAT (β3AR; n = 7, UCP1, PGC1α; n = 6–7, BCAT2, BCKDH; n = 12–14) were examined on day 7 after adenovirus administration. **(H)** VO₂ and RQ (n = 4–5) were measured on day 4 after adenovirus administration. **(I)** NE-induced VO₂ changes of KK-Ay mice (n = 4–6) were measured on day 3 after adenovirus administration. **(J)** Representative histological analysis images of epididymal WAT and BAT (HE staining); **(K)** β2AR, PGC1α, MyoD, BCKDH, and PPARα mRNA expressions in skeletal muscle (β2AR, PGC1α, BCKDH, PPARα; n = 12–13, MyoD; n = 7); and **(L)** circulating phosphatidylcholine levels (n = 7) were examined on day 7 after adenovirus administration. The scale bars indicate 50 μm. The data underlying the graphs shown in the figure can be found in **S1 Data**. Data are presented as means ± SD. *P < 0.05, **P < 0.01 by the unpaired t test. a-MT; a-methyl-D,L-p-tyrosine, methyl ester, hydrochloride; b2AR, b2-adrenergic receptor; b3AR, b3-adrenergic receptor; BAT, brown adipose tissue; BCAT2, branched-chain AA aminotransferase 2; BCKDH, branched-chain ketoacid dehydrogenase; HE, hematoxylin eosin; IR, insulin receptor; NE, norepinephrine; PPAR, peroxisome proliferator-activated receptor; RQ, respiratory quotient; Rspo3, R-spondin3; WAT, white adipose tissue.

Next, we examined whether hepatic Rspo3 expression has any effects on obesity-induced derangements of body composition including adipose tissue and skeletal muscle. Hepatic Rspo3 induction suppressed body weight gain (Fig 3M) as well as reducing epididymal WAT weight (Fig 4D) with increased β3AR, UCP1, and PGC1α expressions (Fig 4E), suggesting increased sympathetic nerve activity to WAT thereby raising WAT-derived thermogenesis, i.e., promotion of the transition from white to beige adipocytes [27,28]. This observation was also supported by the results of norepinephrine (NE) turnover experiments, which revealed that hepatic Rspo3 induction increased the rate of the NE decrease in epididymal WAT after treatment with α-MT, a catecholamine synthesis inhibitor, suggesting the up-regulation of sympathetic stimulation to WAT with resultant increases of both NE production and turnover (Fig 4F). In contrast, β3AR, UCP1, and PGC1α expressions in BAT were all decreased in obese Rspo3-mice (Fig 4G). As indicated by a previous report showing branched-chain AA (BCAA) metabolism to be necessary for BAT-derived thermogenesis [29], BAT gene expressions of BCAA-catabolizing enzymes such as branched-chain AA aminotransferase 2 (BCAT2) and branched-chain ketoacid dehydrogenase (BCKDH) were decreased in obese Rspo3-mice (Fig 4G). Next, to examine the neural activity in the rostral raphe pallidus nucleus (rRPa), containing the putative sympathetic premotor neurons responsible for thermogenesis in BAT, we performed c-fos IF analysis of the rRPa sections of the brains of these obese mice. The IF results indicated that hepatic Rspo3 induction significantly decreases c-fos IF intensity in these areas, suggesting the suppression of sympathetic activity to BAT (S14 Fig).

Furthermore, to explore whether hepatic Rspo3 induction in obese mice exerts an influence on whole-body energy expenditure, we examined VO₂ and RQ, demonstrating decreased VO₂ and decreased RQ in obese Rspo3 mice (Fig 4H). In addition, we examined whether there are any influences on VO₂ after NE treatment in obese Rspo3 mice, and obtained evidence indicating that hepatic Rspo3 induction decreases the degree of NE-induced VO₂ elevation (Fig 4I). Obesity induced endogenous UCP1 expression in BAT and WAT in murine models (S15A and S15B Fig), resulting in increased resting VO₂ as compared to that of lean mice (S15C Fig). While adipocyte size in epididymal WAT was reduced, adipocytes in BAT were enlarged in obese Rspo3-mice, and this was most clearly observed in KK-Ay mice (Fig 4J). These results suggested that Rspo3 induction in the setting of obesity restores the hyperactivated metabolic rates of BAT-derived adaptive thermogenesis against the development of obesity, while it might be possible to activate WAT-derived thermogenesis. According to these effects, hepatic Rspo3 induction in obese states alters the balance of energy source use shifting toward utilization of lipids to a greater degree than carbohydrates, leading to maintenance of whole-body metabolism.

Hepatic Rspo3 induction increased β2-adrenergic receptor (β2AR) and resultant PGC1α expressions in skeletal muscle (Fig 4K) [30], thereby suggesting the restoration of adrenergic signaling, and skeletal muscle quality [31], which had been impaired in the setting of obesity. It also increased MyoD expression in skeletal muscle (Fig 4K), thereby suggesting the restoration of skeletal muscle remodeling and myogenesis [32], impaired in the setting of obesity. As described in a previous report indicating skeletal muscle PGC1α to regulate BCAA metabolism [33], skeletal muscle expression of BCAA-catabolizing enzymes such as BCKDH was increased in obese Rspo3-mice (Fig 4K). These results suggested that hepatic Rspo3 induction regulates the metabolism of BCAAs in remote organs such as skeletal muscle, thereby increasing the effective utilization of BCAAs, which results in maintenance of the physiological functions of skeletal muscle [34]. In addition, hepatic Rspo3 induction also increased PPARα expression in skeletal muscle (Fig 4K), leading to up-regulation of muscle lipid utilization and oxidation by cooperating with PGC1α [35]. A lipid-mediated inter-organ communication system linking the liver and skeletal muscle was previously identified, in which circulating fatty acids such as phosphatidylcholine (18:0/18:1), synthesized via the hepatic PPARδ-dependent de novo lipogenic pathway in the postprandial state, are utilized in skeletal muscle through its PPARα [22]. According to the results obtained by metabolome analysis, hepatic Rspo3 induction increased the levels of choline (1.8-fold, P < 0.01) and phosphorylcholine (2.3-fold, P < 0.01) or ethanolamine phosphate (2.8-fold, P < 0.01) (S8 and S9 Figs), which are substrates for phosphatidylcholine synthesis through the Kennedy (CDP-choline) pathway or the methylation (phosphatidylethanolamine N-methyltransferase (PEMT)) pathway, respectively [36]. These results suggested that hepatic Rspo3 induction increases phosphatidylcholine synthesis in the liver, actually leading to the up-regulation of circulating phosphatidylcholine levels in obese Rspo3 mice (1.24-fold as compared to control mice) (Fig 4L). This raises the possibility that the inter-organ communication system operating under the hepatic PPARδ-phosphatidylcholine-muscle PPARα axis is involved, to a limited extent, in the ameliorations of both fatty liver and reduced skeletal muscle quality in obese Rspo3-mice. Therefore, these results indicated that hepatic Rspo3 induction improved the obesity-associated body composition imbalance between adipose tissue and skeletal muscle, leading to amelioration of the systemic metabolic disturbance.

A neuronal inter-organ communication system is involved in Rspo3-induced remote effects

Next, we focused on the mechanism underlying this inter-organ communication system, in which hepatic Rspo3 induction in obesity improves glucose metabolism and alters body composition by diminishing obesity-related enlargement of WAT and by restoring sustained stimulation of adaptive thermogenesis (UCP1 elevation in BAT) in obesity. To address whether afferent neuronal signals from the liver are involved in these effects, we performed selective hepatic vagotomy (HV) [37] on an obese murine model (KK-Ay mice). There was no difference of body weight gain after sham or HV operations between sham operation (SO)-LacZ and HV-LacZ mice (S16A Fig), indicating that HV alone does not influence body composition, while HV nullified the body weight reduction obtained in response to hepatic Rspo3 induction. As for food intake, hepatic Rspo3 induction decreases the intakes of obese mice following either sham or HV operations (S16B Fig), indicating that HV alone does not influence the Rspo3-derived inhibitory effect on feeding. In obese Rspo3-mice, hepatic expression of Rspo3 and Rspo3-induced liver weight gains were unaffected by HV (Figs 5A and S16C). Hepatic expressions of genes associated with glucose metabolism such as PEPCK and GK (Fig 5B) as well as lipid metabolic enzymes such as FASN and PPARδ (Fig 5C) were essentially unaffected by HV. Rspo3-induced changes in the expressions of genes, such as LGR5, Axin2, Hsd17, and Cyp2f2, were also unaffected by HV (S16D and S16E Fig). In addition, Rspo3-induced alterations of Rspo3 IF pattern in the liver were unaffected by HV (S16F Fig). Under these conditions, HV almost completely blocked Rspo3-induced β3AR, UCP1 and PGC1α expression increases (Fig 5D) as well as Rspo3-induced WAT weight decrease and the reduction of white adipocyte size observed in SO-Rspo3 mice (Fig 5E and 5F). In addition, HV almost completely blocked Rspo3-induced β3AR, UCP1 and PGC1α expression decreases in BAT and the enlargement of brown adipocyte size observed in SO-Rspo3 mice (Fig 5G and 5H). As for BCAA metabolism, HV also almost completely blocked Rspo3-induced BCAT2 and BCKDH expression decreases in BAT (S16G Fig). Focusing on the NE contents in adipose tissues, HV almost completely blocked Rspo3-induced increases in the NE contents of WAT as well as the Rspo3-induced decrease in the NE contents of BAT (Fig 5I). Furthermore, HV almost completely blocked Rspo3-induced PGC1α and PPARα expression increases in skeletal muscle (Fig 5J). These results indicate a neuronal inter-organ communication system consisting of the hepatic vagus and sympathetic nerves to be involved in remote effects on adipose tissues and skeletal muscle, i.e., alterations of body composition, observed in Rspo3 mice.

Fig 5 — **(A–L)** Selective HV or a sham operation (Sham) was performed 7 days prior to the adenovirus administration. Rspo3 (red bars and circles) or LacZ (blue bars and circles) adenovirus was administered to these mice. **(A)** Hepatic Rspo3 expression (n = 5–8), **(B)** hepatic PEPCK and GK expressions (n = 5–8), and **(C)** hepatic FASN and PPARδ expressions (n = 5–8) were examined on day 7 after adenovirus administration. **(D)** β3AR, UCP1, and PGC1α mRNA expressions in epididymal WAT (n = 6–7), **(E)** epididymal WAT weight (n = 4–6), **(F)** representative histological analysis images of epididymal WAT (HE staining), **(G)** β3AR, UCP1, and PGC1α mRNA expressions in BAT (β3AR, UCP1; n = 5–8, PGC1α; n = 10–14), **(H)** representative histological analysis images of BAT (HE staining), **(I)** NE contents in WAT and BAT (n = 6–7), and **(J)** PGC1α and PPARα mRNA expressions in skeletal muscle (n = 7–11) were obtained 7 days after adenovirus administration. The scale bars indicate 50 μm. **(K)** Glucose tolerance and **(L)** insulin tolerance tests were both performed on day 7 after adenovirus administration (n = 5–14). The data underlying the graphs shown in the figure can be found in **S1 Data**. Data are presented as means ± SD. *P < 0.05, **P < 0.01, #P < 0.05, †P < 0.05 by the unpaired t test. b3AR, b3-adrenergic receptor; BAT, brown adipose tissue; FASN, fatty acid synthetase; GK, glucokinase; HE, hematoxylin eosin; HV, hepatic vagotomy; NE, norepinephrine; PEPCK, phosphoenolpyruvate carboxykinase; PPAR, peroxisome proliferator-activated receptor; Rspo3, R-spondin3; WAT, white adipose tissue.

Next, to examine whether HV influences Rspo3-induced improvement of glucose intolerance and insulin resistance in obese KK-Ay mice, we performed glucose tolerance and insulin tolerance tests. In SO mice, Rspo3 induction ameliorated glucose intolerance and insulin resistance in obese mice, but these improvements were partially blocked by HV (Fig 5K and 5L). These results suggested hepatic Rspo3 induction to ameliorate glucose intolerance and insulin resistance via the combination of decreased gluconeogenesis and increased glycolysis locally in the liver and/or improved peripheral insulin sensitivity systemically. While the former mechanisms do not require inter-organ communication mediated by the afferent vagus and efferent sympathetic nerves, the latter does.

Suppression of Rspo3 leads to metabolic derangements promoting obesity development

Next, we examined whether suppression of hepatic Rspo3 exerted any effects on glucose metabolism and body composition. We generated tamoxifen-induced liver-specific Rspo3 knockout mice (iLiRspo3KO mice) by crossing serum albumin promoter-CreER mice and Rspo3-floxed mice obtained employing CRISPR/CAS9 genome editing technology (S17 Fig). Tamoxifen administration at a low dose to these mice decreased hepatic Rspo3 expression by almost 20%, followed by a much greater degree of decrease in Rspo3 protein levels (by almost 48%) (Fig 6A). RNA-Scope revealed that Rspo3-positive signals are also decreased in iLiRspo3KO mice (by 13%) (S18A Fig) though to a milder degree than indicated by the results of quantitative RT-PCR (Fig 6A). Suppression of hepatic Rspo3 to this degree changed neither cumulative food intake nor body weight gain for the 1-week period from day 28 or 4, respectively, after tamoxifen administration (S18B Fig). Under these conditions, hepatic GK expression was locally decreased but hepatic PEPCK expression was unchanged in iLiRspo3KO mice (Fig 6B) with no change in liver weight (S18C Fig). In addition, while hepatic LGR5, a downstream target of the Rspo3/Wnt signaling pathway, as well as Oat, Rdh9, and Cyp7a1, markers of genes in the pericentral zone, were decreased, while hepatic Cyp2f2, a marker of genes in the periportal zone, tended to be increased (S18D and S18E Fig). Suppression of Rspo3 exacerbated glucose intolerance and decreased insulin sensitivity in normal chow-fed iLiRspo3KO mice (Fig 6C and 6D), with these non-beneficial effects persisting 2 months after tamoxifen administration (S18F Fig). The examination of insulin signaling demonstrated that suppression of Rspo3 down-regulated insulin-stimulated serine473 phosphorylation of Akt in remote organs, such as skeletal muscle and epididymal WAT, as well as in the livers of iLiRspo3KO mice (Fig 6E).

As for remote organs, UCP1 expression in WAT was decreased (Fig 6F) with no change in WAT weight (S18G Fig), and UCP1 expression in BAT was increased (Fig 6G). BCAA-catabolizing enzyme (BCKDH) in BAT was also increased in these iLiRspo3KO mice (Fig 6G). These observations are consistent with the results showing suppression of Rspo3 to increase VO₂ and to slightly increase RQ, leading to up-regulation of whole-body energy expenditure (Fig 6H), a phenomenon with consequences opposite those observed in obese Rspo3-mice (Fig 4H). We examined whether suppression of Rspo3 impacts NE-induced VO₂ change, and obtained results indicating that hepatic Rspo3 suppression increases the VO₂ elevation in response to NE treatment (Fig 6I). In addition, β2AR and PGC1α expressions in skeletal muscle were decreased, which in turn decreased skeletal muscle expression of BCKDH (Fig 6J). Suppression of hepatic Rspo3 down-regulates BCAA metabolism in skeletal muscle, thereby diminishing the effective utilization of BCAAs, which results in deterioration of the capacity to maintain skeletal muscle functions [34]. Skeletal muscle expression of PPARα was also decreased in these mice (Fig 6J), a phenomenon with consequences opposite those observed in obese Rspo3-mice (Fig 4K). Therefore, suppression of hepatic Rspo3 is associated with obesity-induced insulin resistance, systemically, as well as diabetes and changes in body compositions including the distributions of adipose tissue and skeletal muscle.

Next, tamoxifen administration at a moderate dose to these mice decreased hepatic Rspo3 expression, by almost 40% (S18H Fig), as well as the number of Rspo3-positive hepatocytes in the pericentral zone (S18I Fig). Under these conditions, while hepatic LGR5 and Axin2, markers of the downstream target of the Rspo3/Wnt signaling pathway, and Cyp7a1, a marker of genes in the pericentral zone, were decreased, hepatic Cyp2f2, a marker of genes in the periportal zone, was increased (S18J Fig). Suppression of Rspo3 increased hepatic SCD1 expression, while decreasing hepatic PPARδ expression (Fig 6K), a phenomenon with consequences opposite those observed in obese Rspo3-mice (Fig 3F and 3J). These results raise the possibility that the inter-organ communication system operating under the control of the hepatic PPARδ-to-muscle PPARα axis is negatively regulated, resulting in decreased fatty acid utilization in skeletal muscle. In addition, suppression of Rspo3 increased lipid droplets, as revealed by Oil red-O staining and calculated TG contents of the liver in normal chow-fed iLiRspo3KO mice, indicating the progression of fatty liver change (Fig 6L and 6M). These results demonstrate a reduction in hepatic Rspo3 expression of only 20% to 40% to exacerbate obesity-associated features, suggesting that Rspo3 may be a key player contributing to the regulation of both hepatic metabolic zonation and systemic glucose metabolism, as well as body composition, via inter-organ communication systems.

Discussion

Metabolic homeostasis of nutrients, in mammals, is maintained via inter-organ communications, which are mediated by humoral secreted factors, the autonomic nervous system and the vascular system, constructing a myriad of networks linking remote organs in the body [38]. Several organs secrete an array of bioactive polypeptides, such as adipokines [39], hepatokines [40], and myokines [41], into the circulation, contributing to maintenance of systemic metabolism. As with these humoral factors, global research attention is now being focused on the roles of neuronal communication systems in this inter-tissue metabolic coordination [42–45]. As for the significance of the blood vascular system [46], suppression of endothelial inflammation or ER stress improves obesity-induced insulin resistance, leading to lifespan prolongation [47,48]. Therefore, most organs in the body function as metabolic sensors for a variety of nutrients and as production sites for many of the important signals, regarding nutritional status, which are transmitted to other organs via humoral factors, neuronal or vascular systems.

Conducting studies under the broad umbrella of global basic sciences, we have identified an inter-organ communication system mediated by a neuronal network originating in the liver and have proposed that this system plays an important role in the maintenance of homeostatic metabolism [38]. In detail, neuronal signals from the liver were recently identified as regulating basal metabolic rates [37], increasing pancreatic β cell mass [49,50], and contributing to obesity-related hypertension [51]. However, in the present age of satiation, obesity develops and disturbs this metabolic homeostasis, resulting in pathophysiological states such as diabetes and metabolic syndrome [52], which are among the highest priority problems, in terms of social and medical economics, faced by the modern world.

In this study, we focused on the spatiotemporal sensing and interacting mechanism(s) of metabolic zonation in the liver. Hepatic landmark genes of metabolism for the different lobular zones were previously determined by both laser microdissection [53] and spatiotemporal transcriptomics studies [54–56]. Thus, metabolic features, under the control of these spatially restricted gene expressions, are considered to be positional markers of the different zones. As for the mechanism(s) underlying this metabolic zonation, the Wnt/β-catenin signaling pathway is a master regulator for gene expressions of the pericentral metabolic enzymes according to the results of studies showing a pericentral-portal gradient in the activity of this signaling pathway [11,57]. This activity gradient is formed by both Wnt and Rspo ligands expressed around central veins and sinusoidal endothelial cells [13,14,58]. However, the precise mechanisms mediating the zonated expression pattern of these ligands and constructing the activity gradient of their downstream signaling pathway has yet to be clarified.

We next investigated the role of Rspo3, a key molecule modulating hepatic zonation via the Wnt/β-catenin signaling pathway [4], by regulating its expression throughout the liver, resulting in the identification of a new inter-organ “neuronal” communication system functioning to maintain metabolic homeostasis. Furthermore, we have strived to elucidate both the pathophysiological significance and the mechanism(s) by which this inter-organ communication system is disturbed during obesity development, thereby contributing to the exacerbation of obesity-associated features.

First, hepatic Rspo3 expression was shown to be enhanced and to converge around central veins in response to feeding as compared to the fasting state (i.e., spatiotemporal characteristics of Rspo3), while being attenuated in obesity, reflecting the physiological and pathophysiological significance of Rspo3.

Second, hepatic Rspo3 induction ameliorates obesity-associated features, such as fatty liver change, diabetes, and insulin resistance including those impacting remote organs. Locally in the liver, hepatic Rspo3 induction, reconstituting metabolic zonation, revealed a novel inter-nutrient mechanism by which the balance between lipid and glucose metabolism is maintained, thereby playing an important role in suppressing obesity-related fatty liver progression (S12C Fig). Outside the liver, hepatic Rspo3 induction ameliorated reduced skeletal muscle quality in obesity by restoring insulin signaling and up-regulating muscle PPARα expression and muscle fatty acid utilization. These remote effects of Rspo3 on skeletal lipid utilization through PPARα suggest the possible involvement of the neuronal communication operating under the hepatic vagus as well as non-neuronal communication operating under the hepatic PPARδ-phosphatidylcholine-muscle PPARα axis [22]. This complicated inter-organ system has been considered to play a significant role in maintaining metabolic homeostasis mainly in the postprandial state, which is compatible with the finding that hepatic Rspo3 converges in the pericentral zones after feeding and exerts several metabolic functions, as described in the present report. In addition, hepatic Rspo3 induction diminished enlargement of WAT in obese mice, while it restored sustained stimulation of BAT-mediated adaptive thermogenesis against obesity development. These remote effects on different adipose tissues were almost completely nullified by selective dissection of the hepatic vagal nerve. The remote effect in WAT is similar to the phenomenon mediated by neuronal signals originating in hepatic PPARγ expression, which modifies white adipocyte characteristics and regulates energy metabolism [37]. However, the finding that hepatic PPARγ expression was changed in lean Rspo3-mice but not necessarily in obese Rspo3-mice suggests the existence of a neuronal mechanism, different from that originating in hepatic PPARγ, which functions in the maintenance of WAT metabolism. The remote effect in BAT is similar to the so-called energy-saving machinery mediated by neuronal signals originating in hepatic GK expression, down-regulating thermogenesis-related genes in BAT, i.e., BAT thermogenic physiology [59]. On the contrary, as observed in the setting of obesity, decreases in hepatic Rspo3 expression and its function contribute to reducing the stimulation signal delivered to WAT-derived thermogenesis, resulting in WAT enlargement, while it contributes to producing that delivered to BAT-derived adaptive thermogenesis against obesity development, a phenomenon which precisely reflects the pathophysiology of obesity. As previously reported, BAT-enriched adipokine (Adissp) serves as a major upstream signaling component especially in inguinal WAT thermogenesis, but not either BAT or epididymal WAT thermogenesis, and improves glucose homeostasis, suggesting the significance of a mechanism that distinguishes and balances thermogenesis derived from different regions of adipose tissues [60]. Our present results raise the possibility that reconstitution of hepatic metabolic zonation by Rspo3 induction in obesity, with increased WAT-derived thermogenesis and reversal of overstimulated BAT-derived thermogenesis, may lead to balancing and redistribution of thermogenesis derived from different adipose tissues such as WAT and BAT. Thus, interpretation of the complex results obtained in this study points to the involvement of a newly-identified “neuronal” inter-organ communication system arising from the control of hepatic metabolic zonation by Rspo3 in the maintenance of thermogenic homeostasis, which differs between adipose tissues such as WAT and BAT, highlighting the novel and diverse roles of neuronal signals transmitted by the hepatic vagal nerve.

Third, selective knockdown of hepatic Rspo3 exacerbates diabetes and fatty liver change, and then decreases insulin sensitivity in remote organs such as skeletal muscle and adipose tissue as well as in the liver. Again, down-regulation of hepatic Rspo3 changes the expressions of genes relevant to adipose tissue and skeletal muscle, i.e., genes related to thermogenesis as well as those related to myogenesis, mitochondrial biogenesis, and lipid utilization, respectively, leading to deterioration of body composition and organ quality.

Taking the present results into consideration, the Rspo3-induced reconstitution of hepatic metabolic zonation, which is disturbed in obesity, plays an important role in regulating local hepatic and systemic metabolism of nutrients and may restore the systemic composition of adipose tissues while improving skeletal muscle quality, by producing a newly identified complicated inter-organ communication system. On the other hand, prolonged obesity disturbs the physiological and metabolic functions originating in the control of hepatic metabolic zonation by Rspo3, leading to the progression of obesity-associated features such as glucose intolerance, insulin resistance, fatty liver, and deterioration of organ quality (Figs 7 and S19).

Fig 7 — Hepatic metabolic zonation mediated by Rspo3 plays an important role in maintaining metabolism in remote organs, such as adipose tissues and skeletal muscles, as well as locally in the liver through the inter-organ coordination consisting of neuronal and non-neuronal communication. However, in the setting of obesity, the impaired Rspo3 function in the liver leads to the disturbance of this Rspo3-mediated physiological inter-organ mechanism, paradoxically resulting in progression of pathophysiological features such as glucose intolerance, insulin resistance, fatty liver, and deterioration of organ quality. BAT, brown adipose tissue; Rspo3, R-spondin3; WAT, white adipose tissue.

A recent study, focusing on liver regeneration, implicated the proliferation signals from hepatocytes in the mid-lobular zones in the repopulation and regeneration of hepatocytes in the neighboring pericentral and periportal zones [61]. Namely, hepatocytes in these different lobular zones, which have distinct characteristics in liver regeneration [62] or metabolism (described in the present study), interact to construct novel inter-cellular or inter-organ communication systems, respectively, contributing to the maintenance of both liver and whole-body homeostasis.

However, a limitation of this study is that we did not strictly examine whether hepatic Rspo3 induction in obesity has effects more prolonged than those observed up to 23 days after adenovirus administration. The detailed mechanism, by which modulation of hepatic Rspo3 expression exerts long-term effects on whole-body metabolism and body composition, via an inter-organ communication system, awaits elucidation and merits further investigation.

In the currently aging society, with rising rates of diabetes, metabolic syndrome and sarcopenia, medical interventions designed to “extend healthy life expectancy” are urgently needed [63]. Recently, new technical tools targeting neuronal inter-organ communication have been developed. The first involves stimulating the hepatoportal nerve plexus with focused ultrasound to restore glucose homeostasis in diabetic animals [64]. The second innovation is stimulating parasympathetic nerves to pancreatic β cells with an optogenetic signal to increase insulin secretion, a strategy applicable to diabetic patients [65]. If an innovative technology to selectively stimulate hepatic Rspo3 or its interacting vagal nerves can be developed, it may open novel avenues to accessing this newly identified inter-organ communication system derived from modulation of hepatic Rspo3-mediated metabolic zonation. Therefore, targeting Rspo3 is very important for achieving therapeutic innovations and applications for patients suffering from diabetes, obesity, and obesity-associated diseases. Such medical breakthroughs are eagerly awaited by the public.

Materials and methods

Animals

Animal studies were conducted in accordance with Teikyo University institutional guidelines. All experimental protocols had been approved by the Institutional Animal Care and Use Committee of the Teikyo University Environmental & Safety Committee (Teikyo University Ethics Committee for Animal Experiments) and assigned numbers as 18–037, 18–038, and 18–040. Male C57BL/6J and C57BL/6J-Lep^ob/ob (ob/ob) mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). C57BL/6J-DIO mice were obtained by 4-week feeding of a high-fat diet (HFD-60, with total calories provided by 62.2% lipids, 19.6% carbohydrates, and 18.2% proteins) (Oriental Yeast Co., Ltd., Tokyo, Japan) beginning at 5 weeks of age. Male KK-Ay mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). Rspo3 floxed mice (Rspo3^flox/flox) with a C57BL/6 background were created by means of CRISPR/CAS9 technology at the Institute of Laboratory Animals, Tokyo Women’s Medical University (Tokyo, Japan), as previously described [66,67]. The CAS9 endonuclease used in this study is EnGen Cas9 NLS, S. pyogenes (#M0646T, New England Biolabs, Ipswich, Massachusetts, United States of America). The CRISPR RNA (crRNA), trans-activating CRISPR RNA (tracrRNA), and single-strand oligo donor DNA (ssODN) were created by Fasmac Co., Ltd. (Kanagawa, Japan). These sequences are described in S1 Text. The loxP sites were inserted both up- and down-stream from the first exon of the Rspo3 genome using CRISPR/CAS9. The serum albumin promoter-CreERT2 (SA-CreER) mice were obtained from Tohoku University (Miyagi, Japan) [68]. To obtain tamoxifen-inducible liver-specific Rspo3 knockout (iLiRspo3KO) mice, we crossed SA-CreER mice and Rspo3^flox/flox mice. At 9 weeks of age, male SA-CreER; Rspo3^flox/flox mice and male Rspo3^flox/flox mice, as controls, were injected intraperitoneally with tamoxifen (#T5648, Sigma, St. Louis, Missouri, USA), at the low dose of 40 μg/g body weight, dissolved in corn oil (#C8267, Sigma) every 24 h for 4 consecutive days (total dose of 160 μg/g body weight). These mice were analyzed 7 and 13 days after 4 tamoxifen administrations. At the moderate dose of 120 μg/g body weight, tamoxifen was injected intraperitoneally to 6-week-old male SA-CreER; Rspo3^flox/flox mice and control mice every 24 h for 3 consecutive days (total dose of 360 μg/g body weight). These mice were analyzed 2 days after 3 tamoxifen administrations. All of these animals were housed in an air-conditioned environment, with a 12-h light-dark cycle. Only male mice were used for these experiments.

Preparation of recombinant adenoviruses

Recombinant adenovirus, containing the murine R-spondin3 (Rspo3) under the control of the CMV promoter, was prepared using the Adeno-X Adenoviral System 3 (#632267, Takara Bio Inc., Otsu, Japan). Rspo3 fragments for constructing the adenovector are described in S1 Text. Recombinant adenovirus containing the bacterial β-galactosidase gene was used as a control [69]. Recombinant adenoviruses were propagated in HEK293 cells and purified employing cesium chloride gradient ultracentrifugation [70]. Titers of these viruses were determined by endpoint cytopathic effect assay.

Administration of recombinant adenoviruses

Recombinant adenoviruses were injected into the tail veins of either standard chow-fed 8-week-old male C57BL/6J, ob/ob, and KK-Ay mice or high fat-fed 8-week-old DIO mice at a dose of 5.0 × 10⁷ plaque forming units as previously reported [37].

Immunoblotting

Tissue samples were homogenized in ice-cold RIPA Lysis buffer containing 1×RIPA Lysis buffer, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and protease inhibitor cocktail at a 1:100 dilution (#sc-24948A, Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA). Tissue homogenates were centrifuged and the supernatants including tissue protein extracts (20 μg total protein) were boiled in Laemmli buffer containing 10 mM dithiothreitol, then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) employing the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc., Hercules, California, USA). The liver samples were immunoblotted with antibodies including anti-Rspo3 antibody (#32155, GeneTex, Inc., Alton Pkwy Irvine, California, USA), anti-PDK4 antibody (#12949, Proteintech Group, Inc., Rosemont, Illinois, USA), anti-PPARδ antibody (#74076, Cell Signaling Technology, Inc., Danvers, Massachusetts, USA), anti-Cpt1a antibody (#114337, GeneTex, Inc.), and anti-GAPDH antibody (#5174, Cell Signaling Technology, Inc.), as previously reported. The immunoblots using antibodies obtained from GeneTex, Inc., Proteintech Group, Inc., and Cell Signaling Technology, Inc. were routinely performed at a 1:2,000 dilution in Can Get Signal (#NKB-101, TOYOBO, Osaka, Japan), followed by secondary antibodies (ECL Anti-Rabbit IgG, horseradish peroxidase (HRP)-linked Whole Antibody (from donkey)) (#NA934V, GE Healthcare, Buckinghamshire, United Kingdom) at a 1:5,000 dilution. The immunoblots were visualized with an ECL Prime Western Blotting System (#GERPN2232, Merck KGaA, Darmstadt, Germany).

Immunoblotting of nuclei

The nuclear fraction was isolated as described in the protocol of Schreiber and colleagues [71] with slight modifications. Tissue samples were homogenized in ice-cold buffer (pH 7.9) containing 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCL, 0.5 mM DTT, 0.05% NP-40, and protease inhibitor (cOmplete ULTRA, mini, EASYpack (#05892970001, Merck KGaA)). Tissue homogenates were centrifuged and the pellets including nuclear protein extracts were dissolved in buffer containing 5 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol, and 0.3 M NaCl. Dissolved pellets were centrifuged and the supernatants were boiled in Laemmli buffer containing 10 mM dithiothreitol, then subjected to SDS-PAGE employing the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc.). The nuclear samples from the liver were immunoblotted with antibodies including anti-Foxo1 antibody (#18592-1-AP, Proteintech Group, Inc.) and anti-HDAC1 antibody (#2062, Cell Signaling Technology, Inc.), as previously reported. The immunoblots using antibodies obtained from Proteintech Group, Inc. and Cell Signaling Technology were routinely performed at a 1:2,000 dilution with Can Get Signal (TOYOBO), followed by secondary antibodies (ECL Anti-Rabbit IgG, HRP-linked Whole Antibody) (#NA934V, GE Healthcare) at a 1:5,000 dilution. The immunoblots were visualized with an ECL Prime Western Blotting System (Merck KGaA).

Blood analysis

Blood glucose levels were measured using the glucose analyzer GLUTESTMINT (SANWA KAGAKU KENKYUSHO CO., LTD., Nagoya, Japan) and serum insulin levels were assayed with the Insulin ELISA Kit (#M1102, Morinaga Institute of Biological Science, Inc., Kanagawa, Japan). Blood Rspo3 levels were measured with the R-Spondin-3 PicoKine ELISA Kit (#EK1525, Boster Biological Technology, Pleasanton, California, USA). Blood phosphatidylcholine levels were measured with the Phosphatidylcholine Assay Kit (#Ab83377, Abcam plc., Cambridge, UK).

Histological analysis

Liver tissue, WAT, and BAT were removed and fixed in 10% formalin and embedded in OCT compounds, then subjected to the bio-tissue freezing slicing method (Leica CM1520 cryostat, Leica Biosystems, Nussloch, Germany). Tissue sections were stained with HE, Oil red-O, and PAS. The images of these stained samples were obtained with an all-in-one Fluorescence Microscope (BZ-X800, KEYENCE, Osaka, Japan) and analyzed using the BZ-X Analyzer software (KEYENCE).

Fluorescent immunostaining analysis of the liver

For formalin fixation, the liver was removed and fixed in 10% formalin. For reperfusion fixation, mice anesthetized with medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg) were perfused transcardially with 4% paraformaldehyde (PFA), and then the liver was removed and fixed in 4% PFA. These liver samples were embedded in OCT compounds, then subjected to the bio-tissue freezing slicing method (Leica Biosystems). Five-μm-thick sections were immunostained with anti-Rspo3 polyclonal rabbit antibody (#32155, GeneTex, Inc.) at a 1:100 dilution as the primary antibody and Cy3 affinity pure donkey anti-rabbit IgG (#711-165-152, Jackson ImmunoResearch Laboratories, Inc., West Grove, Pennsylvania, USA) at a 1:200 dilution as the secondary antibody. In addition, liver sections were immunostained with anti-HNF4α polyclonal rabbit antibody (#bs-3828R, BIOSS Inc., Woburn, Massachusetts, USA), and anti-CK19 polyclonal rabbit antibody (#14965-1-AP, Proteintech Group, Inc.) at a 1:100 dilution [72], and anti-Ki67 rabbit monoclonal antibody (#12202S, Cell Signaling Technology, Inc.) at a 1:200 dilution as the primary antibodies and Cy3 affinity pure donkey anti-rabbit IgG (#711-165-152, Jackson ImmunoResearch Laboratories, Inc.) at a 1:200 dilution as the secondary antibody. Liver sections were immunostained with anti-CD31 rat antibody (#550274, BD Bioscience, Franklin Lakes, New Jersey, USA) at a 1:20 dilution as the primary antibody and Goat Anti-Rat IgG (Cy5) (#ab6565, Abcam plc.) at a 1:100 dilution as the secondary antibody. The nuclei of hepatocytes were visualized with Cellstain DAPI solution (#D523, DOJINDO LABORATORIES, Kumamoto, Japan). The images of these immunostained samples were obtained with an inverted confocal laser scanning microscope (Zeiss Super-Resolution Microscope LSM880 with AiryScan, Carl Zeiss, Oberkochen, Germany) and analyzed using the ZEISS ZEN and Imaris software (Carl Zeiss). The intensity of Rspo3 immunofluorescence was measured on 4 lines, each 20 μm from blood vessel walls along the lines at right angles centered on the hepatic central veins obtained at random, according to the manufacturer’s instructions.

Fluorescent immunostaining analysis of rRPa of the brain

For formalin fixation, the brain was removed and fixed in 10% formalin. These brain samples were embedded in OCT compounds, then subjected to the bio-tissue freezing slicing method (Leica Biosystems). Ten-μm-thick sections were immunostained with anti-c-fos monoclonal rabbit antibody (#2250S, Cell Signaling Technology, Inc.) at a 1:500 dilution as the primary antibody and Cy3 affinity pure donkey anti-rabbit IgG (#711-165-152, Jackson ImmunoResearch Laboratories, Inc.) at a 1:200 dilution as the secondary antibody. The images of these immunostained samples were obtained with an all-in-one Fluorescence Microscope (BZ-X800, KEYENCE) and analyzed using BZ-X Analyzer software (KEYENCE).

RNA-Scope (in situ hybridization)

The liver was removed and fixed in 10% formalin and embedded in OCT compounds, then subjected to the bio-tissue freezing slicing method (Leica Biosystems). Five-μm-thick liver sections were subjected to the hybridization of RNAscope-specific probes. Three probes against mouse Rspo3 (RNAscope Probe-Mm-Rspo3, #402011), mouse Ppib (RNAscope Positive Control Probe-Mm-Ppib, #313911), and DapB (RNAscope Negative Control Probe-DapB, #310043) were prepared employing Advanced Cell Diagnostics, Inc. (Newark, California, USA). We used the single-plex RNAscope assay according to the User Manual of RNAscope 2.5 HD Reagent Kit-Brown (#322300), in which Rspo3-positive signals (as dots) are detected with DAB [73]. Quantification of Rspo3-positive signals in the liver sections is provided by counting the numbers of dots according to the manufacturer’s instructions for the BZ-X Analyzer software (KEYENCE).

Glucose and insulin tolerance tests

Glucose and insulin tolerance tests were performed 5 to 20 days after adenovirus administration or 7 days to 2 months after 4 tamoxifen administrations, as previously described [37]. Insulin (0.15 U/kg of body weight for lean mice and 0.25 U/kg of body weight for obese and SA-CreER; Rspo3^flox/flox mice) was injected into the intraperitoneal space.

Immunoprecipitation

Mice fasted 16 h were injected with 200 μl of normal saline, with or without 10 U/kg body weight of insulin, via the tail vein. The liver, hindlimb muscle, and epididymal WAT were removed 300 s later and then homogenized in ice-cold RIPA Lysis buffer containing 1×RIPA Lysis buffer, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and a protease inhibitor cocktail at a 1:100 dilution (#sc-24948A, Santa Cruz Biotechnology, Inc.). The tissue homogenates were centrifuged and the supernatants including tissue proteins were used for immunoprecipitation with 5 μl of anti-Akt antibody (#9272, Cell Signaling Technology, Inc.) or anti-insulin receptor antibody (#3025, Cell Signaling Technology, Inc.), coupled with nProtein A-Sepharose 4 Fast Flow (#GE17-5280-01, Merck KGaA). Next, the samples immunoprecipitated with anti-Akt antibody were subjected to SDS-PAGE and then immunoblotted using anti-phospho-Akt (Ser473) antibody (#4060, Cell Signaling Technology, Inc.) or anti-Akt antibody at a 1:3,000 dilution in Can Get Signal (TOYOBO), followed by secondary antibodies (ECL Anti-Rabbit IgG, HRP-linked Whole Antibody) (#NA934V, GE Healthcare) at a 1:10,000 dilution, as previously described [37]. The samples immunoprecipitated with anti-insulin receptor antibody were immunoblotted using anti-phospho-Tyrosine antibody (#9411, Cell Signaling Technology, Inc.) or anti-insulin receptor antibody at a 1:3,000 dilution, as described above.

Hepatic glycogen content

Frozen livers were homogenized, and glycogen contents were measured using a Glycogen Colorimetric/Fluorometric Assay Kit (#646–100, BioVision, Inc., Milpitas, California, USA), as previously described [74].

Hepatic TG content

Hepatic TG contents were measured as previously described [37]. Frozen livers were homogenized in the buffer (137 mmol/L NaCl, 20 mmol/L Tris, pH 7.5, 1 mmol/L MgCl₂, 1 mmol/L CaCl₂, and 10% glycerol), followed by extraction with chloroform: methanol (2:1, vol:vol), drying, and resuspension in isopropanol. TG contents were measured using a LabAssay Triglyceride (#632–50991, FUJIFILM Wako, Osaka, Japan).

Adipose NE contents

Adipose tissues were rapidly removed and weighed. Tissue samples were homogenized in 0.4 M perchloric acid and were centrifuged. Supernatant norepinephrine levels were measured commercially by SRL (Tokyo, Japan) [75].

NE turnover

NE turnover was measured on the basis of the decrease in adipose tissue NE contents after the inhibition of catecholamine biosynthesis with α-methyl-D,L-p-tyrosine, methyl ester, hydrochloride (α-MT) (#sc-219470, Santa Cruz Biotechnology, Inc.). At 0 and 4 h after α-MT injection (200 mg/kg, i.p.), the mice were sacrificed by cervical dislocation and adipose tissues were rapidly removed and weighed. Tissue samples were homogenized in 0.4 M perchloric acid and then centrifuged. Supernatant NE levels were measured commercially by SRL (Tokyo, Japan) [75].

Whole-body oxygen consumption and respiratory quotient (RQ)

Whole-body oxygen consumption and RQ were measured by an indirect calorimetric method using the Oxymax system (Equal 4 Chamber/Small Subject System), equipped with a four-chamber airtight metabolic cage, and the data obtained with this system were analyzed using CLAMS software (Columbus Instruments, Columbus, Ohio, USA). Mice, in non-anesthetized states, were acclimated to the individual metabolic cages for 1 day prior to the experiment. Volumes of oxygen consumed (VO₂) and carbon dioxide produced (VCO₂) for each metabolic cage were collected every 10 min for 2 days, with room air as a reference. The RQ was calculated by dividing the VCO₂ by the VO₂, used to estimate the contribution of fats and carbohydrates to whole-body energy metabolism in vivo.

NE-induced whole-body oxygen consumption

For measurement of whole-body O₂ consumption after stimulation with NE, the mice were anesthetized with medetomidine, midazolam, and butorphanol tartrate, and VO₂ measurements were conducted for 30 min to obtain basal values. Mice were then briefly removed from the calorimetry chamber, injected intraperitoneally with 1 mg/kg body weight of L-norepinephrine hydrochloride (#74480, Merck KGaA), and returned to the chamber, followed by VO₂ measurement for another 60 min.

Quantitative RT-PCR–based gene expressions

Total RNAs from the liver, WAT and BAT, and skeletal muscle were purified with miRNeasy Mini Kits (#217004, QIAGEN, Valencia, California, USA). cDNAs were synthesized from total RNA of several tissues with both RNasin Ribonuclease inhibitor (#N2111, Promega Corporation, Madison, Wisconsin, USA) and M-MLV-Reverse-Transcriptase-RNase-H-Minus (#M5301, Promega Corporation). Quantitative RT-PCR was performed by Taqman analysis (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with a real time PCR quantitative system or a Syber Green System, THUNDERBIRD SYBR qPCR Mix (#QPS-201, TOYOBO CO., LTD., Osaka, Japan) with MyGo Pro Real Time PCR (IT-IS Life Science Ltd, Joyce House, Ireland). The relative amount of mRNA was calculated with GAPDH mRNA as the invariant control [76]. The Taqman probes and PCR primers used in this study are presented in S1 Text.

RNA-sequencing of hepatic tissues

RNA-sequence analyses were performed commercially by Cell Innovator Co., Ltd. (Fukuoka, Japan) [77]. GO term analysis was performed by using the web-accessible program DAVID (Database for Annotation, Visualization, and Integrated Discovery), which integrates functional genomic annotations. KEGG pathway analysis was also performed employing the DAVID program.

Metabolomics of hepatic tissues

Metabolomic analysis was performed commercially by Human Metabolome Technologies, Inc. (Yamagata, Japan) [78].

Hepatic vagotomy

Selective HV was performed as previously described [52]. After being anesthetized with an intraperitoneal injection containing a mixture of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg), a laparotomy incision was made on the ventral midline and the abdominal muscle wall was opened with a second incision. The gastrohepatic ligament was severed using fine forceps, and the stomach was gently retracted, revealing the descending ventral esophagus and the ventral subdiaphragmatic vagal trunk. The hepatic branch of this vagal trunk was then transected using fine forceps or surgical scissors.

Quantification and statistical analysis

The individual numerical values underlying the graphs and heatmaps shown in the figures can be found in S1 Data. The representative results derived from multiple experiments were displayed. All data are expressed as means ± SD. The statistical significance of differences was assessed employing the unpaired t test.

Supporting information

S1 Fig. Azan-staining and immunofluorescence analysis.

Representative image of Azan-staining and representative immunofluorescence analysis image with anti-Rspo3 antibody and DAPI staining, of pericentral and periportal zones in the liver. The scale bars indicate 100 μm. (The scale bar in whole picture of the liver specimen indicates 1 mm.)

(TIF)

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S2 Fig. Immunofluorescence analysis of lean and obese mice.

Representative immunofluorescence analysis image with anti-Rspo3 antibody and DAPI staining of pericentral and periportal zones in the liver. The scale bars indicate 100 μm.

(TIF)

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S3 Fig. Hepatic gene profiles induced by Rspo3 in lean mice.

(A–H) Rspo3 (red bars and circles) or LacZ (blue bars and circles) adenovirus was administered to standard diet-fed control mice. (A) Rspo3 mRNA expression in WAT, BAT, and skeletal muscle were examined on day 7 after adenovirus administration (WAT, skeletal muscle; n = 5, BAT; n = 9–10). (B) Hepatic LGR5 and Axin2 mRNA expressions (n = 6–7), (C) hepatic Oat, Cyp7a1, and Rdh9 mRNA expressions (n = 7), (D) hepatic Hsd17 and Cyp2f2 mRNA expressions (n = 6–7) were examined on day 7 after adenovirus administration. (E) Hepatic Ki67 mRNA expressions were examined on day 7 (n = 7) and day 23 (n = 5) after adenovirus administration. (F) Hepatic gene expressions associated with glucose and lipid metabolism were examined on day 23 (n = 5) after adenovirus administration. (G) Glucose tolerance and (H) insulin tolerance (n = 5) tests were performed on day 20 and day 19 after adenovirus administration, respectively. The data underlying the graphs shown in the figure can be found in S1 Data. Data are presented as means ± SD. *P < 0.05, **P < 0.01 by the unpaired t test.

(TIF)

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S4 Fig. Hepatic gene profiles induced by Rspo3 in obese mice.

(A–I) Rspo3 (red bars) or LacZ (blue bars) adenovirus was administered to standard diet-fed ob/ob mice. (A) Rspo3 mRNA expression in WAT, BAT, and skeletal muscle were examined on day 7 after adenovirus administration (n = 10–12). (B) Representative RNA-Scope analysis images of the liver in obese Rspo3 mice with specific Rspo3 probe. The numbers of Rspo3-positive signals (dots) were measured randomly (2 areas per mice, n = 3). (C) Circulating Rspo3 levels were measured on day 3 (n = 6) and 7 (fast, n = 13–14, fed, n = 7) after adenovirus administration. (D) Hepatic LGR5 and Axin2 mRNA expressions (n = 6); (E) hepatic Oat, Cyp7a1, and Rdh9 mRNA expressions (n = 5–6); (F) hepatic Hsd17 and Cyp2f2 mRNA expressions (n = 6) were examined on day 7 after adenovirus administration. (G) Representative immunofluorescence analysis images of the liver in obese Rspo3 mice with anti-Rspo3 antibody and DAPI staining, followed by fluorescence analysis of ZsGreen1, and (H) representative immunofluorescence analysis images of the liver in obese Rspo3 mice with anti-Ki67 antibody and DAPI staining, followed by fluorescence analysis of ZsGreen1, and (I) hepatic Ki67 mRNA expressions (n = 4–5) were examined on day 23 after adenovirus administration. The scale bars indicate 100 μm. The data underlying the graphs shown in the figure can be found in S1 Data. Data are presented as means ± SD. **P < 0.01 by the unpaired t test.

(TIF)

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S5 Fig. Gene ontology (GO) analysis of RNA-sequence of the liver.

RNA-sequence analysis was performed by using mRNA sample of the liver from lean mice and obese (ob/ob) mice (n = 4), followed by GO analysis. The data underlying the graphs shown in the figure can be found in S1 Data.

(TIF)

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S6 Fig. KEGG pathway analysis of RNA-sequence of the liver.

RNA-sequence analysis was performed by using mRNA sample of the liver from lean mice and obese (ob/ob) mice (n = 4), followed by KEGG pathway analysis. The data underlying the graphs shown in the figure can be found in S1 Data.

(TIF)

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S7 Fig. Obesity alters hepatic gene expressions associated with Wnt signaling pathway and those of the pericentral or periportal zones.

Lean (purple bars) or ob/ob (yellow bars) mice were used. (A) Hepatic gene expressions associated with Wnt signaling pathway, (B) those of the pericentral zones, and (C) those of the periportal zones (n = 5) were examined by quantitative RT-PCR. The data underlying the graphs shown in the figure can be found in S1 Data. Data are presented as means ± SD. **P < 0.01 by the unpaired t test.

(TIF)

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S8 Fig. Heatmap of metabolome analysis of the liver.

Rspo3 or LacZ adenovirus was administered to standard diet-fed ob/ob mice. Metabolome analysis was performed by using the liver (n = 4). The data underlying the heatmap shown in the figure can be found in S1 Data.

(TIF)

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S9 Fig. Metabolome analysis of the liver.

Rspo3 (red bars) or LacZ (blue bars) adenovirus was administered to standard diet-fed ob/ob mice. Metabolome analysis was performed by using the liver (n = 4) and graphs were obtained from relative areas. The data underlying the graphs shown in the figure can be found in S1 Data. Data are presented as means ± SD. *P < 0.05, **P < 0.01 by the unpaired t test.

(TIF)

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S10 Fig. Gene ontology (GO) analysis of RNA-sequence of the liver.

Rspo3 or LacZ adenovirus was administered to standard diet-fed ob/ob mice. RNA-sequence analysis was performed by using mRNA sample of the liver (n = 4), followed by GO analysis. The data underlying the graphs shown in the figure can be found in S1 Data.

(TIF)

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S11 Fig. RNA-sequence analysis of the liver.

Rspo3 or LacZ adenovirus was administered to standard diet-fed ob/ob mice. RNA-sequence analysis was performed by using mRNA sample of the liver (n = 4). Heatmaps of genes with glucose and lipid metabolic processes were presented. The data underlying the heatmaps shown in the figure can be found in S1 Data.

(TIF)

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S12 Fig. Rspo3 locally alters obesity-related features in the liver of obese mice.

(A) Insulin-stimulated serine473 phosphorylation of Akt protein in liver is presented. Immunoblotting with anti-phospho-Akt (Ser473) and Akt antibodies. The relative intensities (the ratios of p-Akt/Akt protein volume) were indicated in the p-Akt panel. (B) Hepatic gene expressions associated with glucose and lipid metabolism were examined on day 23 (n = 4–5) after adenovirus administration. (C) The scheme of a hypothesis was presented that hepatic metabolic homeostasis is maintained with a balance between glucose and lipid. The data underlying the graphs shown in the figure can be found in S1 Data. Data are presented as means ± SD. *P < 0.05, **P < 0.01 by the unpaired t test.

(TIF)

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S13 Fig. Rspo3 improves obesity-related insulin resistance.

(A–F) Rspo3 (red circles) or LacZ (blue circles) adenovirus was administered to diet-induced obese (A and B) (n = 6), standard diet-fed KK-Ay (C) mice (n = 6) and standard diet-fed ob/ob (D and E) mice (n = 5). (A and C) Glucose tolerance and (B) insulin tolerance tests were performed on day 7 after adenovirus administration. (D) Glucose tolerance and (E) insulin tolerance tests were performed on day 20 and day 16 after adenovirus administration, respectively (n = 5). (F) Insulin-stimulated serine473 phosphorylation of Akt protein in skeletal muscle (hindlimb muscles) and WAT were immunoblotted with anti-phospho-Akt (Ser473) antibody, or the individual antibody. The relative intensities (the ratios of p-Akt/Akt protein volume) were indicated in the p-Akt panels. The data underlying the graphs shown in the figure can be found in S1 Data. Data are presented as means ± SD. *P < 0.05, **P < 0.01 by the unpaired t test.

(TIF)

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S14 Fig. Rspo3 decreases c-fos IF intensity in rRPa.

Rspo3 or LacZ adenovirus was administered to standard diet-fed KK-Ay mice. Representative images of HE staining and representative immunofluorescence analysis images of rRPa with anti-c-fos antibody and DAPI staining on day 10 after adenovirus administration. The scale bars indicate 100 μm.

(TIF)

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S15 Fig. Obesity increases adaptive thermogenesis.

(A and C) Lean (purple bars) or ob/ob (yellow bars) mice were used. (B) Lean (purple bars) or KK-Ay (yellow bars) mice were used. (A) Endogenous UCP1 mRNA expressions in BAT (n = 7) and epididymal WAT (n = 5), and (C) VO2 and RQ (n = 4–5) were examined in lean and ob/ob mice. (B) Endogenous UCP1 mRNA expressions in BAT (n = 7) and epididymal WAT (n = 6–7) were examined in lean and KK-Ay mice. The data underlying the graphs shown in the figure can be found in S1 Data. Data are presented as means ± SD. *P < 0.05, **P < 0.01 by the unpaired t test.

(TIF)

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S16 Fig. HV does not influence hepatic gene profiles but brocks BAT gene profiles, induced by hepatic Rspo3 induction.

(A–G) Selective hepatic vagotomy (HV) and sham operation (Sham) were performed 7 days prior to the adenovirus administration (Rspo3; red bars and LacZ; blue bars). (A) Body weight gain and (B) food intake in either SO-LacZ or HV-LacZ mice were examined (n = 7–8). (C) Liver weight (n = 5–8), (D) hepatic LGR5 and Axin2 expressions (n = 5–8), (E) hepatic Hsd17 and Cyp2f2 expressions (n = 5–8), (F) representative immunofluorescence analysis images of the liver in SO- or HV-KK-Ay Rspo3 mice with anti-Rspo3 antibody and DAPI staining, and (G) BCAT2 and BCKDH expressions in BAT (n = 6–8) were examined on day 7 after adenovirus administration. The scale bars indicate 100 μm. The data underlying the graphs shown in the figure can be found in S1 Data. Data are presented as means ± SD. *P < 0.05, **P < 0.01 by the unpaired t test.

(TIF)

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S17 Fig. Construction of iLiRspo3KO mice.

The scheme of methods was presented that iLiRspo3KO mice and control mice were created by both CRISPR/CAS9 and Cre-loxP technologies.

(TIF)

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S18 Fig. Hepatic gene profiles and metabolic profiles of iLiRspo3KO mice.

(A–H and J) iLiRspo3KO mice (pink bars and circles) or control mice (bright green bars and circles) were presented. (A) Representative RNA-Scope analysis images of the liver in iLiRspo3KO mice with specific Rspo3 probe. The numbers of Rspo3-positive signals (dots) were measured randomly. (B) Food intake (n = 6) and body weight gain (n = 7–9) for 1 week from day 28 or day 4 after tamoxifen administration, respectively. (C) Liver weights were measured (n = 8–9), (D) hepatic LGR5, and Axin2, (E) Cyp7a1, Rdh9, Oat, and Cyp2f2 mRNA expressions (n = 7–8), and (G) epididymal WAT weight (n = 8–9) were examined on day 13 after tamoxifen (40 μg/g body weight) administration. (F) Glucose tolerance and insulin tolerance tests were performed almost 2 months (n = 6–7) after tamoxifen (40 μg/g body weight) administration. (H) Hepatic Rspo3 expression was examined on day 2 after tamoxifen (120 μg/g body weight) administration (n = 7–8). (I) Representative immunofluorescence analysis images of the pericentral zone in the liver with anti-Rspo3 antibody and DAPI staining. (J) Hepatic LGR5, β-catenin, Axin2, Cyp7a1, and Cyp2f2 mRNA expressions were examined on day 2 after tamoxifen (120 μg/g body weight) administration (n = 6–8). The scale bars indicate 50 μm. The data underlying the graphs shown in the figure can be found in S1 Data. Data are presented as means ± SD. *P < 0.05, **P < 0.01 by the unpaired t test.

(TIF)

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S19 Fig. The scheme of a newly identified inter-organ communication system.

(TIF)

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S1 Table. RNA-Sequence analysis of hepatic metabolic genes in the pericentral zones (PC genes) or those in the periportal zones (PP genes).

(TIF)

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S2 Table. RNA-Sequence analysis of hepatic metabolic genes in the pericentral zones (PC genes) or those in the periportal zones (PP genes).

(TIF)

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S1 Raw Images. Western blot sets used in the article.

(PDF)

pbio.3002955.s022.pdf^{(19.5MB, pdf)}

S1 Data. Raw data supporting Figs 1–6, S3–S13, S15, S16, and S18.

(XLSX)

pbio.3002955.s023.xlsx^{(591KB, xlsx)}

S1 Text. Supporting information about probe, primer, and sequences.

(DOCX)

pbio.3002955.s024.docx^{(35.8KB, docx)}

Acknowledgments

The authors thank colleagues and collaborators for their helpful support and advice; J. Hwang and Y. Tezuka for histological analysis; and K. Murata for highly capable research assistance. Finally, the authors gratefully acknowledge the commitment and generosity of the Teikyo University School of Medicine.

Abbreviations

AA: amino acid
b2AR: b2-adrenergic receptor
b3AR: b3-adrenergic receptor
BAT: brown adipose tissue
BCAA: branched-chain amino acid
BCAT2: branched-chain AA aminotransferase 2
BCKDH: branched-chain ketoacid dehydrogenase
Cpt1a: carnitine palmitoyltransferase 1a
Cyp2f2: cytochrome P450 2f2
Cyp7a1: cholesterol 7alpha-hydroxylase
FASN: fatty acid synthetase
G1P: glucose-1-phosphate
G6P: glucose-6-phosphate
GK: glucokinase
GO: gene ontology
HE: hematoxylin eosin
HNF4a: Hepatocyte nuclear factor 4a
HRP: horseradish peroxidase
Hsd17: hydroxysteroid 17-beta dehydrogenase
HV: hepatic vagotomy
IF: immunofluorescence
IR: insulin receptor
ISH: in situ hybridization
NE: norepinephrine
Oat: ornithine aminotransferase
PAS: periodic acid–Schiff
PEPCK: phosphoenolpyruvate carboxykinase
PFA: paraformaldehyde
PPAR: peroxisome proliferator-activated receptor
Rdh9: retinol dehydrogenase9
RQ: respiratory quotient
rRPa: rostral raphe pallidus nucleus
Rspo3: R-spondin3
SCD1: stearoyl CoA desaturase 1
SO: sham operation
SREBP1c: sterol regulatory element-binding protein 1c
TG: triglyceride
WAT: white adipose tissue

Data Availability

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. The original data for the RNA-sequencing experiments have been deposited on the Gene Expression Omnibus (GEO accession GSE282890). Metabolomics data has been deposited to the Metabolomics Workbench (accession number ST003586).

Funding Statement

The studies described were supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKENHI numbers 18H02858, 22H03130, and 23K24389 to KU). KU has received research grants from Mochida Memorial Foundation for Medical and Pharmaceutical Research, Daiwa Securities Health Foundation, the TERUMO LIFE SCIENCE FOUNDATION, the Takeda Science Foundation, and the Koyanagi Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Biol. doi: 10.1371/journal.pbio.3002955.r001

Decision Letter 0

Lucas Smith

30 Aug 2023

Dear Dr Uno,

Thank you for submitting your manuscript entitled "Rspo3, a mediator of hepatic metabolic zonation, ameliorates diabetes and obesity predisposition via inter-organ communication" for consideration as a Research Article by PLOS Biology.

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PLoS Biol. doi: 10.1371/journal.pbio.3002955.r002

Decision Letter 1

Lucas Smith

25 Oct 2023

Dear Dr Uno,

Thank you for your patience while your manuscript "Rspo3, a mediator of hepatic metabolic zonation, ameliorates diabetes and obesity predisposition via inter-organ communication" was peer-reviewed at PLOS Biology, and I apologize again for the delay in sending you a decision. Your study has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers.

In light of the reviews, which you will find at the end of this email, we would like to invite you to revise the work to thoroughly address the reviewers' reports. As you will see below, the reviewers find the study interesting, but highlight that more work is needed to fully support the conclusions.

Given the extent of revision needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is likely to be sent for further evaluation by all or a subset of the reviewers.

We expect to receive your revised manuscript within 3 months, but in this case, we appreciate that that addressing all of the reviewer concerns may require a considerable effort, and we would be happy to extend the deadline for your revision by a few months, if that is helpful. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

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Lucas Smith, Ph.D.

Senior Editor

PLOS Biology

lsmith@plos.org

------------------------------------

REVIEWS:

Reviewer #1: In their manuscript entitled "Rspo3, a mediator of hepatic metabolic zonation, ameliorates diabetes and obesity predisposition via inter-organ communication" Uno and colleagues highlight neuronal and non-neuronal inter-organ linkages to be involved in hepatic Rspo3-mediated maintenance of systemic glucose metabolism and body composition. Therefore, the authors artificially increase Rspo3 expression using an adenovirus-based delivery system in lean and obese mouse models. They show that increased Rspo3 expression ameliorates obesity-associated features, reduces adipose tissue enlargement and restores insulin sensitivity and skeletal muscle quality. By performing a hepatic vagotomy the authors provide some evidence that the effects of Rspo3 include a partial neuronal involvement (ie ameliorated glucose intolerance). Moreover, they identify the hepatic PPARd-to-muscle PPARa-axis to be involved in the regulation of fatty liver development and reduced skeletal muscle quality in obese mice. In line, the authors find these phenotypes to be exacerbated upon reduced hepatic Rspo3 expression.

Overall, Uno et al. highlight an interesting concept linking hepatic WNT signaling which controls liver zonation to the body`s overall health status via inter-organ communication. These findings have the potential to help identifying new therapeutic targets for treating obesity phenotypes. However, several shortcomings of the manuscript should be addressed before publication.

Major comments:

1. The authors claim that Rspo3 expression induced by adenovirus delivery is selective to the liver. However, they never proof this selectivity, and many adenoviral systems are not exclusively targeting the liver. It is a must, to assess Rspo3 expression in other organs (especially adipose tissue and skeletal muscle) to make sure that all observed effects are truly mediated via the liver as claimed by the authors.

2. The authors suggest RSPO3 is mainly expressed in zone3 hepatocytes, whereas many previous studies collectively suggest that WNT ligands, including RSPO3, are mostly expressed in central vein endothelial cells and neighboring sinusoidal endothelial cells. The authors should provide better staining evidence with co-stainings for hepatocyte markers and endothelial cell markers, as well as ISH stainings for RSPO3 with cell type co-stainings to clarify this point. The relatively low reduction in RSPO3 levels in the AlbCreER-induced RSPO3 KO further suggests that the majority of RSPO3 is generated elsewhere.

3. On page 11, lines 7-10 the authors describe upregulation of zone3 and reduction of zone1 metabolic enzymes upon RSPO3 overexpression. This finding is not novel since the role of RSPO3 in metabolic zonation (Rocha et al, Cell Reports 2015) and reprogramming of zone1 into zone3 hepatocytes by RSPO treatment, or deletion of ZNRF3/RNF43, (Sun et al, Cell Stem Cell 2021) have been previously shown. The authors should end that section by referencing these previous findings. Although the authors find exciting new data suggesting RSPO3-related interesting extrahepatic phenotypes, impaired metabolic zonation in patients is major challenge for such a therapy and should be discussed in detail.

4. The authors write in the last sentence on page 11 that RSPO3 slightly increased liver weight, whereas Figure 2E show almost doubled liver weight. RSPO injections increased hepatocyte proliferation and liver weight (Planas-Paz et al, Nature Cell Biology 2016 and Sun et al, Cell Stem Cell 2021) and combined of ZNRF3/RNF43 resulted into uncontrolled hepatocyte proliferation and hepatic tumor formation (Sun et al, Cell Stem Cell 2021). The author have to assess proliferation in their model by Ki67 staining, or if possible by EdU incorporation. Please note the Adenoviral injections induce inflammation which can mask the proliferative effect, so later timepoints (min. 2 weeks post injection) should be assessed. Importantly, the authors have to discuss this caveat associated with potential RSPO3-related therapies.

5. The authors observed that many metabolic genes are deregulated upon increased Rspo3 expression. Instead of picking single candidate genes, they should use their RNA-seq data to get an overall picture (perform GSEA or GO term analysis) and support their hypotheses on the regulation of entire pathways. Complete lists of PC and PP metabolic genes should be shown as well in line with the previous comment.

6. In Fig3A they claim that Rspo3 expression via adenoviral administration may be lower in obese than in lean mice. This comparison is not shown, thus the authors should either add a corresponding graph or remove this sentence.

7. Fig S4 shows a metabolomics analysis. Although the overview map may be helpful to establish the connections between metabolites for the reader, it is more important to convey the message which pathways are up/downregulated. Therefore, the authors should consider exchanging/adding a heatmap instead and group metabolites by pathway. Also here, metabolic processes related to PP hepatocytes should be shown to check whether RSPO3 expression impaired PP hepatocyte metabolism via reprogramming of PP hepatocytes into PC hepatocytes.

8. Fig3D should highlight Akt phosphorylation. However, loading controls and quantification are missing (same for Fig3E, as well as for many other WB analyses!). This must be added for all WB. Additionally, Foxo1 translocation can only be claimed when cytoplasmic levels are shown together with nuclear levels. Otherwise, it might be just increased expression. In addition, the authors conclusions must be toned down (decreased gluconeogenesis and glucose utilization in TCA are not shown by the authors).

9. In Fig S5 the authors make assumptions about minor changes of PPARd and CPT1a (lower than 1.5-fold, and not even confirmed on protein levels). If this remains the only evidence, the authors must tone down their conclusions.

10. The sentence after citation 30" thereby leading to the restoration of adrenergic signaling, mitochondrial biogenesis and skeletal muscle mass [31], which had been impaired in the setting of obesity" is an overstatement considering that the authors only refer to increased b2AR expression and resultant PGC1a expression in the muscle. In general, the entire following sections contains a lot of discussion and correlative findings (i.e. referring to PC18:0/18:1). Either this should be moved into the discussion part, or alternatively, the authors should add some more supportive data here.

11. The authors claim that HV also almost completely blocked Rspo3-induced BCAT2 and BCKDH expression. However, for FigS8D significances are not shown when comparing Rspo3 conditions between Sham and HV as done for literally every other gene. Given the error bars for BCAT, this raises doubts regarding the drawn conclusion. Can the authors show the graph displaying single samples at least to the reviewer?

Minor comments:

1. Most histological staining pictures are too small, quality and size should be increased. CVs and PVs should be labelled.

2. The word "ironically" must be removed from the abstract.

3. What do the authors mean by the term "feeding behavior"? The exact timepoint of analysis after feeding should be stated in the text.

4. The authors use the term "Rspo3 supplementation" throughout the text. However, supplementation is rather used in a dietary context and should be exchanged for a more appropriate term (eg use increased Rspo3 expression).

5. The term "as previous report indicating" used several times throughout the text is grammatically incorrect.

6. Quantification for number of lipid droplets is missing (Fig.3H).

7. FigS5 heatmap should be adjusted to color-blind standards.

8. The graphical abstract in Fig7 is too complicated and should be reduced to the major findings. Alternatively, this extended version could be kept in supplement and a "reduced" version presented in the main figures.

Reviewer #2, Costas Christodoulides (note, Reviewer 2 has signed this review): In this paper, Uno and colleagues investigate the role of hepatic Rspo3 on systemic metabolism using gain- and loss-of-function approaches. Consistent with previous reports, they show that adenovirally mediated, hepatic over-expression of Rspo3 alters liver metabolic zonation (PMID: 26655896) and it is further associated with reduced weight gain and improvements in glucose tolerance and systemic insulin sensitivity in both lean and ob/ob mice. In ob/ob mice, hepatic Rspo3 over-expression also ameliorated hepatic steatosis. In another mouse model of genetic obesity (KK-Ay), hepatic vagotomy appeared to partially reverse the beneficial effects of hepatic Rspo3 over-expression on adiposity and glucose tolerance. Reciprocally, tamoxifen-induced, hepatic knockdown of Rspo3 led to impaired glucose tolerance and systemic insulin sensitivity, as well as, increased hepatic steatosis in chow-fed animals. Based on these findings, the authors postulate that "targeting hepatic metabolic zonation by Rspo3 may provide new therapeutic strategies against diabetes and the metabolic syndrome".

Strengths of the manuscript include that many of the findings are novel and are supported by evidence from both gain- and loss-of-function experiments conducted in vivo and ex vivo. Nonetheless, I have several comments/criticisms as listed below:

Major criticisms:

1. The authors examine only the short-term effects (one week) of hepatic Rspo3 gain- and loss-of-function on adiposity and systemic metabolism (see also comment 6).

2. Based on gene expression analyses, the hepatic over-expression of Rspo3 achieved with adenoviral transductions was very high (10-20x) and was associated with a significant increase in liver size e.g., almost doubling in chow-fed animals (fig. 2e).

3. The authors show in figure 1, that Rspo3 circulates in plasma and furthermore, that it's circulating levels are up-regulated in the fed state and dysregulated in obesity. Yet, they fail to show any data on circulating Rspo3 levels in their gain- and loss-of-function animal models. Additionally, they fail to consider that any of the effects of hepatic Rspo3 on weight gain and systemic metabolism could be mediated by endocrine actions of Rspo3, which I find quite puzzling.

4. Another major limitation, is that the authors failed to measure food intake and energy expenditure in any of their mouse models. This makes it difficult to interpret and draw firm conclusions from their findings.

5. The abstract and results sections should be factual. Yet, they contain a lot of speculation. For example, the authors claim that a remote, lipid mediated, liver-muscle communication system mediates some of the beneficial effects of hepatic Rspo3 on systemic metabolism and improves skeletal muscle quality. However, the data presented fail to directly demonstrate such an axis. Specifically, the authors do not demonstrate either increased circulating phosphatidylcholine levels in their Rspo3 gain-of-function animal models, or direct effects of phosphatidylcholine on skeletal muscle gene expression, or that their animals have e.g., increased exercise tolerance. Thus these comments are best included as speculation in the discussion. There are multiple other similar examples in the results section.

6. The authors do not provide any direct evidence that the activity of the sympathetic nervous system in adipose tissue is altered in response to changes in hepatic Rspo3 expression, yet this is one of their main conclusions.

7. Rspo over-activation has been linked with cancer e.g., RSPO3 gene fusions which are associated with increased RSPO3 expression have been identified in colon cancer patients (PMID: 34663878). Consistently, transgenic mice with increased circulating Rspo levels displayed massive intestinal enlargement in mice (PMID: 16109882, 16357527). On the other hand, Rspo are important for stem cell maintenance and proliferation (PMID: 22617424). Hence, it is highly likely that manipulation of hepatic Rspo3 expression for metabolic disease prevention/treatment purposes will have a very narrow therapeutic window (if at all possible).

Specific points:

1. Could energy repartitioning to the growing liver, and potentially growing intestines, contribute to the beneficial effects of hepatic Rspo3 over-expression on systemic metabolism?

2. The authors claim that "Rspo3 supplementation restores the hyperactivated metabolic rates of BAT-derived adaptive thermogenesis against the development of obesity". However, they do not provide any evidence that BAT is overactive in their ob/ob mice. Secondly, they do not examine any direct effects of Rspo3 on brown adipocyte gene expression. Finally, it is difficult to believe that reduced BAT activity contributes to the reduced weight gain and improved systemic metabolism of mice with hepatic over-expression of Rspo3.

3. I would like to see the effects of hepatic vagotomy on animal weights in figure 5. It is also curious that figure 5D shows data on the ratio of epiWAT/body weight in the vagotomised animals whereas figures 2I and 4D compare the effects of hepatic Rspo3 over-expression on epi WAT weight uncorrected for total body weight.

4. Did hepatic vagotomy have any effects on WAT Ucp1 and Pgc1a gene expression or on systematic insulin sensitivity as assessed by insulin tolerance tests?

5. Did Rspo3 knockdown have any effects on liver size, weight gain, or epididymal WAT weight?

6. Ideally, the authors should quantify the hepatitic lipid content in animals with Rspo3 knockdown and over-expression.

Other points:

1. In figure 1C, was Rspo3 expression reduced in BAT and skeletal muscle from obese mice?

2. Is beta-catenin a WNT target gene?

3. With reference to the data in figure 3E, does PDK4 primarily act in the nucleus?

Reviewer #3: This manuscript by Uno et al reports the role of Rspo3 that is known to determine hepatic zonation in regulating glucose and energy homeostasis. They show that Rspo3 increases with feeding and this rise is attenuated with insulin resistance such as in ob/ob mice. Histologically, they show that Rspo3 is further concentrated in the periportal area and this does not occur in models of insulin resistance. Further, in metabolic tissues such as the liver or adipose tissue. Rspo gene expression progressively declines with insulin resistance such as with high fat diet feeing or in ob/ob mice. Next, they deliver Rspo3 via adenovirus and show increased expression in the liver in addition to improved metabolic parameters such as fasting insulin and glucose levels as well as gluconeogenic genes. This is done also in ob/ob mice showing similar metabolic benefit 7 days post gene delivery. They then examine the mice with deletion of Rspo3 in liver and show metabolic exacerbation. Similar to the overexpression data vi adenovirus, deletion model is also examined after a short term of 7 and 13 days post deletion. Again, this is a short duration given the chronic nature of the diseases being examined and longer term data would be required. Overall, the data presented are interesting and they use multiple in vivo models as well as overexpression and deletion models to show the importance of Rspo3 in metabolic regulation. However, throughout the manuscript, the data interpretation is far-reaching and many of their statements are not at all supported by the data presented as follows. It is suggested that the authors more accurately describe the data without overreaching with conjecture. In particular, much of what is shown as a working model in Fig 7 is not at all shown in their data and would strongly suggest revising to only show what is well-supported by their data.

What is the difference between data presented in Fig 1B versus 1C, and why are the data different for the liver shown in B versus C?

Fig 1E needs to be shown in a lower magnification that demonstrate the zonation more clearly. The image shown in the figure does not capture the peri-central zonation convincingly.

Fig 2A shows increased Rspo3 levels in gene and protein expression. The increase in protein is unclear from the western blot shown and should be quantified. The degree of increase in gene versus protein expression is not at all congruent and expression in other tissues need to be shown. Furthermore, histologic imaging needs to be shown to test whether the increased expression of Rspo3 is more enriched pericentrally as shown with endogenous Rspo3.

What is the evidence that 'hepatic Rspo3 induction exerted remote effects, i.e., the weight of epididymal WAT was significantly decreased (Fig 2I),' - they don't have data to support this statement. The effect in the adipose tissue may be from its expression in this tissue and not the distant effect of the liver, especially since they've already shown expression of Rspo3 in adipose tissue (Fig 1C). Also, in this same section, their postulation involving the sympathetic activation is completely not supported by their data and should be removed.

All the metabolic experiments are done 7 days post gene delivery. Given the chronic nature of the metabolic conditions such as diabetes and obesity that they are investigating, more longer term experiments need to be done as well as gene dosage effect. How did they come up with the dose of gene to be delivered. What are the expression levels of the gene delivery in other tissues such as the adipose tissue, muscle, brain or the immune cells? These are all tissues that can affect the metabolic outcome.

Similar comments re duration after knockout apply as stated above. 7 and 13 days post knockout is not sufficient to gain insight into the chronic nature of the diseases being examined.

For knockdown experiments, why only 20%? This needs to be explained. Is this because more knockdown would lead to severe hepatic derangements? This is an important issue that needs to be addressed and shown with more significant knockdown.

There are multiple inappropriate descriptions of their data or of the subject matter that they describe as follows:

Abstract - pg 4 - Rspo3 is not a 'bioactive substance' - they have manipulated gene expression using adenovirus to investigate its role - this needs to be accurately described in the abstract and throughout the manuscript.

When describing data in Fig 1D, they should be insulin 'responsive' tissue - , not insulin 'reactive'. Also, it's not feeding 'behaviour'. It's simply 'feeding' that would lead to metabolic abnormalities.

'Hepatic Rspo3 supplementation' would not be the best way to describe their experiment. Supplementation has a nutrition connotation and should simply describe their systemic gene delivery rather than 'supplementation'. This is used throughout the section describing findings for Fig 3, which is advised to be removed.

'hepatic PPARd-to-muscle PPARa axis' - again is overstretch of data interpretation.

PLoS Biol. 2025 Jan 24;23(1):e3002955. doi: 10.1371/journal.pbio.3002955.r003

Author response to Decision Letter 1

26 Apr 2024

Attachment

Submitted filename: Responses.docx

pbio.3002955.s025.docx^{(71.2KB, docx)}

PLoS Biol. doi: 10.1371/journal.pbio.3002955.r004

Decision Letter 2

Lucas Smith

10 Jun 2024

Dear Dr Uno,

Thank you for your patience while we considered your revised manuscript "Rspo3, a mediator of hepatic metabolic zonation, ameliorates diabetes and obesity predisposition via inter-organ communication" for publication as a Research Article at PLOS Biology. Your revised study has been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewers.

As you will see below, the reviewers agree that the study has been strengthened in this revision and reviewer 1 has suggested we accept the study. However, both reviewers 2 and 3 have a number of important lingering concerns that we think should be addressed, including with new data, in another revision before we can consider your study for publication.

We do note that the tone of reviewer 3's comments is quite critical, but after discussing his/her comments with the other reviewers and the Academic Editor, we agree with the substance of many of the concerns raised by this reviewer, including that a number of conclusions have been over-interpreted and are not fully supported. In many places we think that it may be possible to address reviewer 3's comments via textual changes - by toning down some conclusions, reworking the abstract, and adding additional explicit discussions of limitations. We would not require the generation of additional long-term metabolic phenotyping of mice with hepatic RSPO3 expression, as we think that this is beyond the scope of the current work, and there is still value in the current findings without that data. But we think the absence of this data should be discussed as a limitation.

With that said, we also think that new data and analyses will be required to address a number of the concerns raised by Reviewer 2 and 3. While we tend not to invite a second experimental revision, given our and the reviewer interest in the study, we are willing to do so here.

We expect to receive your revised manuscript within 3 months. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may withdraw it.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

*Re-submission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this re-submission checklist: https://plos.io/Biology_Checklist

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

*Blot and Gel Data Policy*

*Protocols deposition*

Sincerely,

Luke

Lucas Smith, Ph.D.

Senior Editor

PLOS Biology

lsmith@plos.org

------------------------------------

REVIEWS:

Reviewer #1, Jan Tchorz (note Reviewer 1 has signed this review): The authors addressed all my comments and should be congratulated to an excellent paper that will certainly get significant attention. Jan Tchorz

Reviewer #2, Costas Christodoulides (note, Reviewer 2 has signed this review): The authors have adequately addressed most of my concerns. However, I do have a few additional comments.

Major Comments:

1. The authors state that modulating hepatic RSPO3 expression may "restore metabolic zonation." Is there any evidence, such as transcriptomic data either generated by the authors or publicly available, that hepatic zonation is disrupted in mouse models of dietary and genetically induced obesity?

2. Effects of Vagotomy: The authors claim that a neural inter-organ communication system is involved in hepatic RSPO3-induced actions in adipose tissues and skeletal muscle. However, according to the data in Supplementary Figure 12A, vagotomy abrogates the weight loss associated with hepatic RSPO3 overexpression in genetically obese mice. Could this be the primary reason for the gene expression changes in adipose tissues and skeletal muscle, as well as the changes in glucose tolerance and insulin sensitivity in hepatic RSPO3-overexpressing mice post-vagotomy? Also, I would like to see the effects of hepatic vagotomy on fasting glucose levels. Is it possible that hepatic vagotomy increases hepatic glucose output, thereby potentially contributing to a switch from fat to glucose metabolism in peripheral tissues in vagotomised RSPO3-expressing mice?

3. In Figure 5, panels D, E, G, and J, please also include data from control LacZ mice.

Other Comments:

1. Hepatic RSPO3 expression in lean animals approximately leads to a doubling of liver weight. This is not a slight change as stated by the authors (page 12, second from bottom line).

2. Duration of Metabolic Benefits: Additionally, 20 days is not a "relatively long period" and should not be reported as such by the authors. They should simply state that metabolic benefits lasted up to 20 days.

3. Page 13, bottom of the second paragraph. The authors state that increased beta3AR, UCP1, and PGC1a expression in epididymal WAT contributes to decreased body weight after 7 days of adenoviral treatment. This is speculation. We are not shown any calorimetry data to support this. In fact, based on the authors' data, the weight loss in these animals is driven by reduced appetite.

4. Page 16, second paragraph. The authors mention that hepatic RSPO3 induction regulates hepatic lipogenesis, but the only support for this is changes in gene expression of lipogenic genes. Hence, this statement should be tempered.

5. Page 20, top paragraph: "This so-called energy-saving machinery in obese …. with regulating BAT thermogenic physiology." This is speculation, hence this statement should be tempered and ideally moved to the discussion.

6. Page 20, bottom paragraph: "Hepatic RSPO3 induction…. is impaired in the setting of obesity." Again, this is speculation as the authors have not shown that adrenergic signaling, mitochondrial biogenesis, skeletal muscle mass, and muscle remodeling are improved consequent to hepatic RSPO3 expression. They just show gene expression changes.

7. In Figure 5I, the effects of vagotomy on norepinephrine content in WAT seem to be driven by an increased content in the LacZ mice rather than a decrease in the mice with hepatic RSPO3 overexpression.

8. The immunofluorescence images are of poor quality, and I suggest using higher resolution figures in any publication.

9. The authors claim that Figure 1C shows that RSPO3 expression is reduced in BAT and skeletal muscle of obese mice. However, the bar charts seem to suggest the opposite.

10. Beta-catenin is post-transcriptionally, rather than transcriptionally, regulated by WNT/beta-catenin signaling. Unlike Axin2 and LGR5, it is not a beta-catenin target gene, and the references quoted by the authors do not support such a premise.

Reviewer #3: This revised manuscript shows some improvement and the authors have attended to some concerns raised by the reviewers. They improved on systematically showing expression patterns of Rspo3 in the liver under various metabolic conditions and in response to feeling and fasting. They have used several models of obesity to show the importance of RSPO3 with overexpression and knockout. However, the study continues to have major concerns around their interpretation of data and overreaching of conclusions that are often stated even before data description in the results section. Major issue is around the acute short-term analysis for a disease model that is long term that limits the significance of the study. They have expanded only some models to 21 days which is still insufficient and also, would be important to show in a longitudinal fashion so that we can get some sense of continual/plateau versus temporary effect.

The following are some specific comments:

Abstract -

The metabolic zonation in the liver is thereby broadly defined as 'spatiotemporal'

variability… it is not spaciotemporal - rather it's a 'spatial' phenomenon - but not temporal - thus the term zonation. This elaboration is not needed in the abstract in particular.

'Herein, we demonstrate that altered hepatic metabolic zonation due to Rspo3 contributes to

maintaining systemic glucose metabolism and body composition via a new mechanism

involving neuronal and non-neuronal inter-organ linkages' - this also is not a statement that should appear in the abstract before data description - pls remove and consider adding at the end if supported by overall data.

Similarly - the following sentence are not data description but rather conclusions. 'Hepatic Rspo3 induction in obesity improves obesity-associated features, e.g., fatty liver, insulin resistance and diabetes, by reversing adipose tissue enlargement and restoring attenuated organ insulin sensitivities, overstimulated adaptive thermogenesis and decreased skeletal muscle quality.' - It would be best to describe data.

The entire abstract is unacceptably composed of interpretation/conjecture rather than data description - would be best to describe/summarize data followed by overall conclusion.

Pg 7 - high-fat diet(s) - should be high fat diet

Fig 1 - Nice work showing systematic data beginning with expression pattern in models of obesity and in lean conditions both fasted and fed. However, to conclude this section with "Therefore, targeting hepatic Rspo3 may allow new mechanisms involved in regulating

metabolic homeostasis in the liver to be identified, which could in turn lead to the

detection of remote effects on systemic metabolism with physiological (against obesity)

or pathophysiological (promoting obesity) significance. Pg 11' is not at all appropriate and would be better to summarize their data.

'we expressed Rspo3' on pg 11 should be 'overexpressed'

For fig 2A 2nd panel Y-axis label - should be 'relative intensity'?

For fig 2 - they show effects after 7 days and day 20 is only shown in S3 without corresponding weight data and also no description of this data in the results section. Body weight data needs to be shown in a longitudinal fashion with absolute weights rather than relative change. Metabolic expression data shown in fig 2 after 7 days also needs to be shown after 20 days - (genes shown in fig 2D).

Fig 2A does not show 'selective transgene expression in the liver' pg 11. For this statement to be supported, they need to show expression in other tissues.

'These results indicated hepatic Rspo3 induction to up-regulate the Rspo3/Wnt signaling pathway in the liver and thereby shifted metabolic gene expressions, a process by which hepatic features of metabolism are considered to move from the periportal to the pericentral zone. These phenomena, as described above, are compatible with those outlined in previous reports which indicated the roles of Rspo3 in regulating metabolic zonation [13] or reprograming of zonation in the liver [18]. (pg 12) is not supported by their data preceding this conclusion.

To examine proliferative capacity of the liver in response to Rspo3 overexpression, Ki67 should be shown histologically. It is not clear what the proliferative impact of Rspo3 overexpression is, particularly given the known impact in malignancy. This might explain the increase in liver weight.

Fig 3 - using ob/ob - again - only examined after 7 days with minimal difference in body weight - absolute body weight in a longitudinal fashion need to be shown for the two groups rather than a relative change as shown in Fig 3M.

'Rspo3 induction increased Ki67 IF activity (S4H and S4I Fig)' (pg 15) is not true-. IF does not measure activity.

p-Akt in the livers of ob/ob mice (Fig 3D) needs to be quantified and need more n. n=2 with intensity shown above that ranges widely and is not sufficient.

These improvements of glucose 'metabolism' (pg 18) - should be 'tolerance'

murine models (S11A and S11B Fig), resulting increased resting VO2 as compared to that

of lean mice (S11C Fig) - should also include once overexpressing Rspo3. Otherwise these data are not helpful for their manuscript.

'This so-called energy-saving machinery in obese Rspo3-mice is partly explained by hepatic GK up-regulation, in which hepatic GK mediates an inter-organ linkage associated with regulating BAT thermogenic physiology [30]. These results raise the possibility that the reconstitution of hepatic metabolic zonation by Rspo3 induction in obesity, with up-regulation of WAT-derived

thermogenesis and restoration of overstimulated BAT-derived thermogenesis, leads to

balancing and redistribution of thermogenesis derived from different adipose tissues such

as WAT and BAT.' (pg 20) - would advise removing from results section. These are all expected findings with protection from weight gain. To implicate adrenergic nerve involvement etc is a complete overstretch. Would advise involving a scientist in metabolism to properly interpret these results.

Implication of vagal nerve in RSPO3 action is not at all clear nor with good rationale. Furthermore, the data presented are with large error bars and not meaningful. HV is not at al helpful in the results and adds to the confusion and distracts the main story. Would suggest removing. It reflects the naïve understanding of their data.

The scheme shown in S15 is not at all supported by their data and reflects very poorly on their understanding of their data. Would advise removing any neuronal involvement and keeping purely the data in the different tissues that are known effects of improvement in hepatic insulin sensitivity.

For the KO data, decrease of less than 20% expression with such phenotype is highly questionable and hard to explain. Also, their IF data (S14I) does not show deletion to any appreciable degree. Also, short term data bring limited clinical relevance with no body weight data.

PLoS Biol. 2025 Jan 24;23(1):e3002955. doi: 10.1371/journal.pbio.3002955.r005

Author response to Decision Letter 2

6 Sep 2024

Attachment

Submitted filename: Responses.docx

pbio.3002955.s026.docx^{(73.3KB, docx)}

PLoS Biol. doi: 10.1371/journal.pbio.3002955.r006

Decision Letter 3

Suzanne De Bruijn

6 Nov 2024

Dear Dr Uno,

Thank you for your patience while we considered your revised manuscript "Rspo3, a mediator of hepatic metabolic zonation, ameliorates diabetes and obesity predisposition via inter-organ communication" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and one of the original reviewers. I apologize for the unusual delay incurred while we attempted to obtain additional advice from the reviewers.

Based on the reviews and on our Academic Editor's assessment of your revision, we are likely to accept this manuscript for publication, provided you satisfactorily address the following data and other policy-related requests.

IMPORTANT:

1) Please change your title to: "Rspo3-mediated metabolic liver zonation regulates systemic glucose metabolism and body mass in mice"

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4) Please modify your abstract to include that this work has been performed in mice. We would also like to suggest you to further edit the abstract, to include more details on the experiments that are performed. We suggest "in this study, we analyse the function of Rspo3 in mice"

-change 'expression patterns of endogenous Rspo3' to 'Rspo3 expression analysis showed that Rspo3 expression patterns are spatiotemporally controlled'

-change 'Hepatic Rspo3 induction in obesity' to 'We find that viral-mediated induction of Rspo3 in hepatic tissue'

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5) please ensure that the transcriptomics and metabolomic data are deposited in public repositories and provide us with the DOI.

6) Please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:

Fig. 1A-D, G, I; Fig. 2A-E, G-M; Fig. 3A-G, I-N; Fig. 4A, B, D-I, K, L; Fig. 5A-E, G, I-L; Fig. 6A-D, F-K, M; Fig. S3A-H; Fig. S4A-F, I; Fig. S5; Fig. S6; Fig. S7A-C; Fig. S8; Fig. S10; Fig. S11; Fig. S12B; Fig. S13A-E; Fig. S15A-C; Fig. S16A-E, G and Fig. S18A-G, H, J

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Reviewer remarks:

Reviewer #2: The authors have adequately addressed my comments.

PLoS Biol. 2025 Jan 24;23(1):e3002955. doi: 10.1371/journal.pbio.3002955.r007

Author response to Decision Letter 3

16 Nov 2024

Attachment

Submitted filename: Responses.docx

pbio.3002955.s027.docx^{(72.5KB, docx)}

PLoS Biol. doi: 10.1371/journal.pbio.3002955.r008

Decision Letter 4

Lucas Smith

27 Nov 2024

Dear Kenji,

Thank you for the submission of your revised Research Article "Rspo3-mediated metabolic liver zonation regulates systemic glucose metabolism and body mass in mice" for publication in PLOS Biology, and thank you for addressing our last editorial requests in this revision. On behalf of my colleagues and the Academic Editor, Jason W. Locasale, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

**IMPORTANT: Please note, that before accepting your study I have updated the data availability statement (DAS) and the supplementary western blot data file, after discussing these with you over email.

The DAS now reads:

"All relevant data are within the paper and its Supporting Information files. The original data for the RNA-sequencing experiments have been deposited on the Gene Expression Omnibus (GEO accession GSE282890). Metabolomics data has been deposited to the Metabolomics Workbench (accession number ST003586).

Further requests for resources and reagents should be directed to and will be fulfilled by the corresponding author, Kenji Uno."

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Azan-staining and immunofluorescence analysis.

(TIF)

pbio.3002955.s001.tif^{(14.5MB, tif)}

S2 Fig. Immunofluorescence analysis of lean and obese mice.

Representative immunofluorescence analysis image with anti-Rspo3 antibody and DAPI staining of pericentral and periportal zones in the liver. The scale bars indicate 100 μm.

(TIF)

pbio.3002955.s002.tif^{(14.4MB, tif)}

S3 Fig. Hepatic gene profiles induced by Rspo3 in lean mice.

(TIF)

pbio.3002955.s003.tif^{(2.4MB, tif)}

S4 Fig. Hepatic gene profiles induced by Rspo3 in obese mice.

(TIF)

pbio.3002955.s004.tif^{(7.4MB, tif)}

S5 Fig. Gene ontology (GO) analysis of RNA-sequence of the liver.

(TIF)

pbio.3002955.s005.tif^{(3.4MB, tif)}

S6 Fig. KEGG pathway analysis of RNA-sequence of the liver.

(TIF)

pbio.3002955.s006.tif^{(4MB, tif)}

S7 Fig. Obesity alters hepatic gene expressions associated with Wnt signaling pathway and those of the pericentral or periportal zones.

(TIF)

pbio.3002955.s007.tif^{(1.7MB, tif)}

S8 Fig. Heatmap of metabolome analysis of the liver.

(TIF)

pbio.3002955.s008.tif^{(5MB, tif)}

S9 Fig. Metabolome analysis of the liver.

(TIF)

pbio.3002955.s009.tif^{(8.1MB, tif)}

S10 Fig. Gene ontology (GO) analysis of RNA-sequence of the liver.

(TIF)

pbio.3002955.s010.tif^{(3.5MB, tif)}

S11 Fig. RNA-sequence analysis of the liver.

(TIF)

pbio.3002955.s011.tif^{(6.2MB, tif)}

S12 Fig. Rspo3 locally alters obesity-related features in the liver of obese mice.

(TIF)

pbio.3002955.s012.tif^{(2.1MB, tif)}

S13 Fig. Rspo3 improves obesity-related insulin resistance.

(TIF)

pbio.3002955.s013.tif^{(2.7MB, tif)}

S14 Fig. Rspo3 decreases c-fos IF intensity in rRPa.

(TIF)

pbio.3002955.s014.tif^{(9.8MB, tif)}

S15 Fig. Obesity increases adaptive thermogenesis.

(TIF)

pbio.3002955.s015.tif^{(1.7MB, tif)}

S16 Fig. HV does not influence hepatic gene profiles but brocks BAT gene profiles, induced by hepatic Rspo3 induction.

(TIF)

pbio.3002955.s016.tif^{(4.9MB, tif)}

S17 Fig. Construction of iLiRspo3KO mice.

The scheme of methods was presented that iLiRspo3KO mice and control mice were created by both CRISPR/CAS9 and Cre-loxP technologies.

(TIF)

pbio.3002955.s017.tif^{(2.1MB, tif)}

S18 Fig. Hepatic gene profiles and metabolic profiles of iLiRspo3KO mice.

(TIF)

pbio.3002955.s018.tif^{(5.5MB, tif)}

S19 Fig. The scheme of a newly identified inter-organ communication system.

(TIF)

pbio.3002955.s019.tif^{(5.4MB, tif)}

S1 Table. RNA-Sequence analysis of hepatic metabolic genes in the pericentral zones (PC genes) or those in the periportal zones (PP genes).

(TIF)

pbio.3002955.s020.tif^{(1.8MB, tif)}

S2 Table. RNA-Sequence analysis of hepatic metabolic genes in the pericentral zones (PC genes) or those in the periportal zones (PP genes).

(TIF)

pbio.3002955.s021.tif^{(1.8MB, tif)}

S1 Raw Images. Western blot sets used in the article.

(PDF)

pbio.3002955.s022.pdf^{(19.5MB, pdf)}

S1 Data. Raw data supporting Figs 1–6, S3–S13, S15, S16, and S18.

(XLSX)

pbio.3002955.s023.xlsx^{(591KB, xlsx)}

S1 Text. Supporting information about probe, primer, and sequences.

(DOCX)

pbio.3002955.s024.docx^{(35.8KB, docx)}

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Data Availability Statement

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PERMALINK

Rspo3-mediated metabolic liver zonation regulates systemic glucose metabolism and body mass in mice

Kenji Uno

Takuya Uchino

Takashi Suzuki

Yohei Sayama

Naoki Edo

Kiyoko Uno-Eder

Koji Morita

Toshio Ishikawa

Miho Koizumi

Hiroaki Honda

Hideki Katagiri

Kazuhisa Tsukamoto

Roles

Abstract

Introduction

Results

Metabolic functions of Rspo3 in the liver are disturbed in obesity

Fig 1. Metabolic functions of Rspo3 in the liver are disturbed in obesity.

Rspo3 influences systemic glucose metabolism and body composition

Fig 2. Rspo3 influences systemic glucose metabolism and body composition.

Rspo3 improves obesity-induced metabolic disorders locally in the liver

Fig 3. Rspo3 improves obesity-induced metabolic disorders locally in the liver.

Rspo3 ameliorates the systemic glucose metabolism and body composition abnormalities in obese mice

Fig 4. Rspo3 ameliorates the systemic glucose metabolism and body composition abnormalities in obese mice.

A neuronal inter-organ communication system is involved in Rspo3-induced remote effects

Fig 5. A neuronal inter-organ communication system is involved in Rspo3-induced remote effects.

Suppression of Rspo3 leads to metabolic derangements promoting obesity development

Fig 6. Suppression of Rspo3 leads to metabolic derangements promoting obesity development.

Discussion

Fig 7. The scheme of a newly identified inter-organ communication system.

Materials and methods

Animals

Preparation of recombinant adenoviruses

Administration of recombinant adenoviruses

Immunoblotting

Immunoblotting of nuclei

Blood analysis

Histological analysis

Fluorescent immunostaining analysis of the liver

Fluorescent immunostaining analysis of rRPa of the brain

RNA-Scope (in situ hybridization)

Glucose and insulin tolerance tests

Immunoprecipitation

Hepatic glycogen content

Hepatic TG content

Adipose NE contents

NE turnover

Whole-body oxygen consumption and respiratory quotient (RQ)

NE-induced whole-body oxygen consumption

Quantitative RT-PCR–based gene expressions

RNA-sequencing of hepatic tissues

Metabolomics of hepatic tissues

Hepatic vagotomy

Quantification and statistical analysis

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Lucas Smith

Roles

Decision Letter 1

Lucas Smith

Roles

Author response to Decision Letter 1

Decision Letter 2

Lucas Smith

Roles

Author response to Decision Letter 2

Decision Letter 3

Suzanne De Bruijn

Roles

Author response to Decision Letter 3

Decision Letter 4

Lucas Smith

Roles