GPR146 in adipose tissue drives adipose-liver crosstalk and promotes hepatic steatosis in mice

Domain: chemistry
Published: 2026-03-03
Editorial outcome: major_revision

Abstract
The limited therapeutic options for metabolic dysfunction-associated steatotic liver disease (MASLD) underscore the need for deeper mechanistic insight and new treatment strategies. Here, we identify the orphan G protein-coupled receptor GPR146 as a regulator of hepatic steatosis through adipose–liver crosstalk. Human genetic analyses link the GPR146 locus to circulating markers of liver injury and inflammation. In mice, both constitutive and acute GPR146 depletion protect against diet-induced obesity and hepatic steatosis. Notably, adipose-specific, but not liver-specific, GPR146 deletion reduces hepatic lipid accumulation by limiting free fatty acid (FFA) influx. Mechanistically, GPR146 promotes adipogenesis in preadipocytes via Gαq-PKC-AKT signaling, increasing lipid storage capacity, and enhances lipolysis in mature adipocytes through ERK activation, elevating circulating FFA. Together, these coordinated actions increase FFA delivery to the liver, promoting triglyceride accumulation. Our findings establish GPR146 as a pleiotropic regulator of adipose tissue biology and a potential therapeutic target for MASLD.

Introduction
Metabolic dysfunction-associated steatotic liver disease (MASLD) and its sequalae are emerging health issues, affecting ~25% of the adult population worldwide1. MASLD comprises a spectrum of hepatic manifestations ranging from the initial steatosis to the more aggressive Metabolic dysfunction-associated steatohepatitis (MASH). MASH is characterized by chronic inflammation and development of fibrosis, which may progress further to cirrhosis, liver failure or hepatocellular carcinoma, resulting a higher demand for liver transplantation2,3. Although multiple therapeutic strategies targeting different pathological processes of MASH, including lipid accumulation, inflammation, and fibrosis, are under clinical trials, only two pharmacologic treatment, Resmetirom, a thyroid hormone receptor- agonist, and Semaglutide, a GLP-1 receptor agonist, have been approved by the FDA for clinical use 4-6. This highlights the urgent need to gain deeper understanding of pathogenesis of MASH for the future development of new therapies.

It is now well recognized that obesity and obesity related metabolic disorders such as Type2 Diabetes Mellitus (T2DM) are major risk factors for MASLD and MASH7. In populations with obesity, the prevalence of MASLD varies from 60% to 95%7,8. A key pathological feature linking obesity to MASLD is adipose tissue dysfunction. While adipose tissue serves as the primary lipid reservoir and plays a crucial endocrine role in systemic energy balance, chronic nutrient overload can lead to a breakdown in adipose expandability, promoting lipolysis, inflammation, and release of free fatty acids (FFAs) into circulation. These excess FFAs are delivered to the liver, where they contribute to hepatic triglyceride accumulation, a hallmark of steatosis, and create a pro-inflammatory environment that promotes progression toward fibrosis9.

Among the molecular pathways orchestrating adipose tissue function, G protein–coupled receptors (GPCRs) play a central role in regulating lipid metabolism, adipogenesis, and systemic energy balance10. Notably, GPCRs, such as the cannabinoid receptor CB1, have been shown to promote hepatic steatosis by increasing adipocyte lipogenesis and FFAs released into circulation. Conversely, adipose-specific deletion of CB1 protects against hepatic lipid accumulation9,11. These findings highlight the essential role of GPCR-mediated adipose signaling in coordinating systemic lipid flux and position adipose-resident GPCRs as important contributors to MASLD pathophysiology.

Building on this concept, we previously identified the orphan GPR146 as a regulator of hypercholesterolemia and atherosclerosis12. In the current study, we extend our investigation to explore the role of GPR146 in the development of obesity and MASLD. We demonstrated that both constitutive and AAV-mediated acute depletion of GPR146 protected mice from diet–induced obesity and hepatic steatosis. These metabolic benefits were sex-specific, with energy expenditure elevated in both male and female knockout mice. The effect was more pronounced in females, driven by increased UCP1-mediated thermogenesis, whereas the mechanism in males remains less clear. To delineate the tissue-specific contributions, we generated adipose- and liver-specific knockout models. Our results show that the reduction in hepatic steatosis, diet-induced obesity and circulating FFA is primarily attributed to GPR146 deficiency in adipose tissue, which limits free fatty acid flux to the liver. Mechanistically, GPR146 promotes adipogenesis through Gαq-PKC-AKT signaling in preadipocytes and enhances lipolytic activity in mature adipocytes via ERK signaling. Together, these findings highlight a critical role for adipose GPR146 in mediating adipose-liver crosstalk and support its potential as a therapeutic target for obesity-related liver disease.

Results
GPR146 locus is genetically associated with markers of liver injury and inflammation

Previous GWAS studies have identified the association between GPR146 and plasma cholesterol levels12-15. Subsequent analysis using the UK Biobank dataset not only confirmed this finding but also revealed an association between the causal SNP in the GPR146 locus, rs1997243, and C-reactive protein (CRP) levels, as well as plasma liver enzymes, including gamma glutamyl-transferase (GGT), aspartate transaminase (AST), and alkaline phosphatase (ALP)16,17. Consistently, meta-analysis of GWAS studies from Common Metabolic Diseases Knowledge Portal18 shows that rs1997243-G allele is linked to increased plasma levels of LDL-C, CRP, GGT, ALP, AST, and alanine aminotransferase (ALT) in humans (Supplementary Fig. 1a, b). In addition, rs1997243-G is associated with an increased expression of GPR146 in human blood and white adipose tissue based on eQTL studies using 670 human blood samples and 591 human subcutaneous adipose tissue samples from GTEx database (Supplementary Fig. 1c, d). Collectively, these findings suggest that GPR146 may play a role in regulating liver function in humans.

GPR146 promotes diet-induced lipid accumulation and inflammatory signaling in Liver

Hepatic injury and inflammation can be triggered by various factors, including metabolic disorders such as MASLD. In some cases, MASLD can progress to a more severe form known as MASH, characterized by liver inflammation and varying degrees of fibrosis1. In this study, we utilized Gpr146 whole-body knockout (Gpr146-/-) and floxed (Gpr146fl/fl) mouse models (Supplementary Fig.2a-c) to investigate the role of GPR146 in the development of MASLD and its associated hepatic injury.

In line with human genetic findings linking elevated GPR146 expression to increased liver enzymes indicative of liver injury, we found that Gpr146 deficiency markedly reduced hepatic triglycerides (TG) contents in both male and female mice fed a high fat diet (HFD) (Fig. 1a, b, d, e). Additionally, serum levels of ALT were significantly decreased in GPR146-deficient mice compared to control littermates (Fig. 1c, f). Moreover, the liver weight of male GPR146-deficient mice was lower than that of their wild-type control littermates when fed HFD, whereas no significant difference was observed in female mice (Supplementary Fig. 2d).

To gain deeper insights into the underlying metabolic alterations, we performed untargeted metabolomic analysis on liver from HFD fed Gpr146+/+ and Gpr146-/- mice. The most notable differences were observed in metabolites related to lipid, amino acids, and glucose metabolism. Among common lipid species, triacylglycerols (TAG) and diacylglycerols (DAG) levels exhibited the most significant reduction, followed by a trend of decreased monoacylglycrols (MAG) and lysophosphatidylethanolamine (LPE) due to GPR146 deficiency (Fig. 1g, h). Additionally, several hepatic free fatty acid (FFA) and oxidized lipid species were significantly diminished in the livers of GPR146-deficient mice (Fig. 1i). Many of these metabolites, such as palmitate, linoleate, 13-hydroxyl-octadecadienoic acid (13-HODE), and dimethylguanidino valeric acid (DMGV), have previously been reported to be upregulated in liver of MASLD patients19,20. Consistently, total hepatic FFA levels were significantly reduced in HFD fed GPR146-deficient mice compared to control littermates (Fig. 1j). Aside from assessing hepatic FFA levels, we also measured their concentrations in plasma. We found that both male and female GPR146-deficient mice exhibited significantly reduced circulating FFA levels compared to control littermates (Fig. 1k).

To investigate the transcriptional basis for the observed phenotypes, we performed transcriptomic analysis of liver tissue from 16h-fasted HFD-fed Gpr146-/- and wild-type littermates. Gene set enrichment analysis (GSEA) revealed that differentially expressed genes (DEGs) were significantly enriched in pathways related to apoptosis, inflammation, and immune response (Fig. 1l, m). Notably, multiple immune-related pathways, including TNFα signaling via NF-κB and interferon responses, were significantly downregulated in Gpr146-/- livers, in line with the reduced hepatic steatosis and lower circulating ALT levels (Fig. 1l, m).

In addition to downregulation of injury and inflammation-related pathways, GSEA also revealed suppression of adipogenesis-related pathways in the liver of GPR146-deficient mice, suggesting that GPR146 may promote hepatic steatosis via PPARγ-mediated transcriptional programs (Fig. 1l, m). Adipogenic hepatic steatosis has been previously linked to overexpression of PPARγ21,22,23,24. In line with these reports, we found that hepatic Pparg expression was drastically elevated by HFD feeding and significantly reduced in GPR146-deficient mice at both mRNA and protein levels (Supplementary Fig. 2e, f, g, h).

Downstream targets of PPARγ involved in lipid droplet formation and lipid uptake, including Cidea, Plin4, Cidec, Cd36, were also significantly downregulated in the livers of Gpr146-/- mice (Supplementary Fig. 2h). Moreover, genes associated with inflammation and fibrosis (Tgfb1, Tgfbi, Col1a1, Col1a2), and extracellular matrix (ECM) remodeling (Dcn, Bgn, Ecm1, Lum) were likewise diminished, suggesting a broader suppression of MASH-associated transcriptional programs in the absence of GPR146 (Supplementary Fig. 2h). Although pathways of de novo lipogenesis and fatty acid oxidation were not significantly enriched in the transcriptomic datasets, qPCR analysis revealed modest but consistent increases in lipogenic genes (Fasn, Acaca, Elovl6) and reductions in fatty acid oxidation genes (Cpt1a, Acadl) in GPR146-deficient livers (Supplementary Fig. 2i). These changes likely represent compensatory responses to the overall reduction in hepatic lipid content. Together, these findings reveal that global GPR146 deficiency leads to suppression of hepatic PPARγ signaling and its downstream adipogenic targets, along with reduced expression of inflammatory and ECM remodeling genes. While these changes are consistent with the reduced hepatic lipid accumulation observed in Gpr146-/- mice, it remains unclear whether suppression of the PPARγ pathway is a driving mechanism or a secondary adaptation to the decreased hepatic triglyceride content.

In summary, through integrated lipidomic, transcriptomic, and histological analyses, our data demonstrate that GPR146 deficiency robustly protects against diet-induced hepatic steatosis. These protective effects are associated with reduced hepatic and circulating free fatty acid levels, downregulation of inflammatory and immune response pathways, and suppression of hepatic PPARγ signaling.

GPR146 deficiency reprograms hepatic glucose and amino acid metabolism

Beyond reduced hepatic lipid accumulation, GPR146 deficiency led to significantly elevated levels of hexose metabolites within the pentose phosphate pathway (PPP), including hexose monophosphate and erythrose-4-phosphate (Supplementary Fig. 3a). These changes suggest a shift in glucose metabolism toward the PPP, potentially increasing NADPH production to maintain redox homeostasis25. We also observed elevated liver glycogen content in both male and female Gpr146-/- mice on chow diet, and in male knockout mice on HFD (Supplementary Fig. 3b, c). Although it remains unclear whether the glycogen accumulation reflects increased glucose uptake or reduced oxidation, these findings suggest a shift in hepatic carbohydrate flux toward storage and anabolic processing, potentially reflecting enhanced hepatic insulin sensitivity in the absence of GPR146.

Strikingly, untargeted metabolomics also revealed significantly elevated hepatic levels of seven standard amino acids in Gpr146-/- mice, namely histidine, cysteine, lysine, valine, leucine, isoleucine, and phenylalanine (Supplementary Fig. 3d). This pattern suggests altered amino acid handling, potentially reflecting reduced catabolism or diminished routing into gluconeogenic and lipogenic pathways26.

Consistent with these hepatic metabolic changes, chow-fed Gpr146-/- mice exhibited a significantly higher respiratory exchange ratio (RER) during the dark cycle compared to controls (Supplementary Fig. 3e, f), indicating a greater reliance on glucose as an energy source. Furthermore, chow-fed Gpr146-/- mice displayed significantly increased lean mass without changes in fat mass (Supplementary Fig. 3g, h), potentially reflecting altered systemic nutrient partitioning and increased availability of amino acids.

In summary, GPR146 deficiency promotes a coordinated metabolic reprogramming of hepatic and systemic metabolism. These findings point to a coordinated shift in hepatic nutrient partitioning that may contribute to the reduced lipid accumulation in the absence of GPR146.

GPR146 Deficiency Protects against Diet-induced Obesity in Mice

MASLD is often accompanied with obesity and insulin resistance1. Starting from 3 weeks of HFD feeding, male Gpr146-/- mice exhibited significantly lower body weight as compared to wild-type control littermates, and this weight divergence continued to increase over time (Fig. 2a). Likewise, the body weight difference between female Gpr146-/- and Gpr146+/+ mice became discernable from 6 weeks after HFD feeding and was more pronounced as HFD feeding continued (Fig. 2e). After three months of HFD feeding, magnetic resonance imaging (MRI) analysis revealed that Gpr146-/- male mice had significant less fat mass than wild type controls (Fig. 2b). Both male and female GPR146-deficient mice exhibited reduced weights of inguinal (iWAT) and epididymal (eWAT) adipose tissue in comparison to their wild type controls (Fig. 2c, d, f). In male Gpr146-/- mice, the interscapular brown adipose tissue (BAT) was also significantly smaller, as evidenced by a reduction in depot weight (Fig. 2d). Histological analyses further showed that adipocytes in both eWAT and iWAT were markedly smaller in Gpr146-/- mice (Fig. 2g, h).

In parallel with the observed reduction in adipose depot mass and adipocyte size, transcriptomic profiling of adipose tissue revealed significant metabolic reprogramming in Gpr146-/- mice. Gene set enrichment analysis (GSEA) showed upregulation of pathways related to fatty acid oxidation, the TCA cycle, and respiratory electron transport in both epididymal (eWAT) and inguinal (iWAT) fat depots, but not in brown adipose tissue (BAT) (Supplementary Fig. 4a). These changes likely reflect enhanced mitochondrial oxidative capacity and increased fatty acid utilization within adipocytes, thereby influence systemic FFA dynamics. Concurrently, immune-related pathways were downregulated (Supplementary Fig. 4b, c, d), accompanied by reduced expression of macrophage markers, including Cd68 and Itgam across all three adipose depots (Supplementary Fig. 4a). Consistent with these transcriptomic changes, histological analysis showed a marked reduction in crown-like structures (CLS) in the eWAT of Gpr146-/- mice, indicating reduced macrophage infiltration and attenuated adipose tissue inflammation (Fig. 2i, j).

We next assessed whether GPR146 deficiency influences systemic glucose metabolism. In male Gpr146-/- mice fed HFD, glucose tolerance was significantly improved compared to control littermates, with differences becoming most apparent during the late phase of the glucose tolerance test (≥ 60 minutes), suggesting enhanced glucose clearance capacity (Fig. 2k). Similarly, insulin tolerance tests showed divergence between genotypes primarily during the late recovery phase from insulin-induced hypoglycaemia (≥ 30 minutes), rather than during the initial insulin response (Fig. 2l and Supplementary Fig. 5b). This temporal pattern-where genotype difference emerges primarily during the late phase of glucose and insulin tolerance tests- suggests that GPR146 deficiency may enhances hepatic insulin sensitivity or modulate counter-regulatory responses, rather than directly improving peripheral insulin sensitivity and glucose disposal. Supporting this interpretation, fasting glucose, plasma insulin levels, and HOMA-IR were comparable between Gpr146-/- and Gpr146+/+ animals (Supplementary Fig. 5c, 5d, 5e).

In summary, GPR146 deficiency confers protection against diet-induced obesity, as demonstrated by reduced fat mass, smaller adipocyte size, and lower circulating free fatty acid levels. These phenotypes are accompanied by enhanced oxidative metabolism and reduced adipose tissue inflammation, highlighting a critical role for GPR146 in promoting white adipose tissue expansion and metabolic dysfunction under conditions of nutritional excess.

GPR146-deficient mice exhibit elevated energy expenditure

To investigate whether altered energy utilization contributes to the lean phenotype of Gpr146-/- mice, and to distinguish potential causal mechanisms from secondary adaptations, we performed indirect calorimetry27,28 after 2 weeks of HFD feeding, a time point when fat mass differences between Gpr146-/- mice and their wild-type littermates begin to emerge (Fig. 3a). Food intake was comparable between genotypes (Fig.3b, c), ruling out the possibility of excessive dietary TG contributing to the observed obesity and hepatic steatosis phenotype. Notably, male GPR146-deficient mice exhibited significantly elevated energy expenditure only during the light/inactive phase, indicating that the lack of GPR146 primarily affects resting energy homeostasis (Fig. 3d, e, Supplementary Fig. 6a-d). In contrast, female knockout mice exhibited elevated energy expenditure throughout the dark/active phase (Fig. 3g, h), indicating a potentially distinct regulatory mechanism compared to males.

The increase in energy expenditure was not attributable to changes in locomotor activity, as Gpr146+/+ and Gpr146-/- littermates exhibited comparable activity levels (Fig. 3f). To explore potential mechanisms underlying the elevated energy expenditure observed in male knockout mice during the light cycle, we examined the expression of genes associated with three major thermogenic pathways: UCP1-mediated uncoupling, creatine cycling, and calcium cycling. No significant changes were detected in the expression of classical thermogenic genes (Ucp1, Dio2, Prdm16), creatine metabolism genes (Ckmt1, Ckmt2), or calcium cycling regulators (Atp2a2, Ryr1, Ryr2) in BAT, eWAT, or iWAT (Supplementary Fig. 6e-g). These results suggest that the increased energy expenditure in male Gpr146-/- mice is not driven by transcriptional activation of known thermogenic programs and may involve alternative or post-transcriptional mechanisms.

In contrast, female Gpr146-/- mice showed robust activation of classical thermogenic program. Specifically, Ucp1 expression increased more than 15-fold in eWAT, but not in iWAT, along with significant upregulation of Pgc1a and Ppara (Supplementary Fig. 6h, i). These findings suggest that, in female Gpr146-/- mice, UCP-1 mediated thermogenesis in eWAT likely contributes to the elevated energy expenditure.

Taken together, our findings suggest that the reduced body weight and adiposity observed in GPR146-deficient mice are likely due to an increase in energy expenditure. In females, this is associated with transcriptional activation of UCP1-dependent thermogenesis. In males, however, the mechanism underlying elevated energy expenditure remains unclear. This emerging sex difference in thermogenic regulation may reflect distinct hormonal influences on adipose tissue plasticity, highlighting the need for further investigation into sex-specific pathways mediating GPR146-dependent metabolic control.

Adipose GPR146, rather than hepatic GPR146, mediates protection against diet-induced obesity and liver steatosis

To determine the tissue origin responsible for the reduced hepatic lipid accumulation observed in Gpr146-/- mice, we first generated liver-specific Gpr146 knockout mice (Alb-Cre+ Gpr146fl/fl mice) and challenged them with HFD feeding. Surprisingly, liver-specific GPR146 depletion did not reduce hepatic TG content or improve metabolic parameters. In fact, male liver-specific knockout mice exhibited increased liver weight (Fig. 4a, b) and a trend of increased TG content (Fig. 4c), while female knockouts displayed significantly increased body weight under HFD feeding (Supplementary Fig. 7a). Notably, circulating FFA levels remained unchanged in both sexes (Fig. 4e, f), further distinguishing the liver-specific phenotypes from whole-body GPR146 deficiency.

Given the lack of phenotype in liver-specific knockouts, we examined tissue-specific expression patterns of GPR146. Based on the Human Protein ATLAS dataset29 (Human Protein Atlas proteinatlas.org) and our own analysis, GPR146 is most abundantly expressed in mature adipocytes within white adipose tissue (Supplementary Fig. 8a, b). Following 8 weeks of HFD feeding, Gpr146 expression decreased significantly in eWAT but remained stable in iWAT and EBAT (Supplementary Fig. 8c). This pattern was confirmed by independent datasets30,31 (Supplementary Fig. 8d, e). Furthermore, transcriptomic meta-analysis of human adipose tissue from the Adipose Tissue Knowledge Portal32 revealed consistent downregulation of GPR146 expression in individuals with obesity compared to lean controls (Supplementary Fig. 8f).

To test the functional role of adipose GPR146, we generated adipose-specific Gpr146 knockout mice (Adipoq-Cre+ Gpr146fl/fl mice) and control littermates (Adipoq-Cre- Gpr146fl/fl mice) and subjected them to HFD challenge. Compared to control littermates, adipose-specific knockout mice exhibited significantly lower liver weight and less hepatic TG content (Fig. 4a-d). In contrast to the liver-specific knockout model, Adipoq-Cre+ mice showed downregulation of hepatic PPARγ target genes and ECM-associated genes (Fig. 4g), paralleling the expression profile seen in whole-body knockouts. Both male and female adipose-specific knockout mice displayed significantly lower body weight and fat mass in response to HFD challenge (Fig. 4h-j and Supplementary Fig. 7b). In males, both iWAT and BAT, but not eWAT were significantly reduced (Fig. 4k). The reduced iWAT mass was accompanied by a decrease in adipocyte size, indicating a mitigation of hypertrophy expansion (Fig. 4l). In female mice, the weight of both eWAT and iWAT depots were reduced in Adipoq-Cre+ mice (Fig. 4m). Importantly, plasma FFA levels were significantly reduced in both sexes (Fig. 4e, f), supporting a role for adipose GPR146 in regulating systemic FFA homeostasis.

Despite these improvements, adipose-specific deletion of Gpr146 only partially recapitulated the phenotypes observed in whole-body knockout mice. For example, male adipose-specific knockout mice exhibited a 22% reduction in hepatic TG (Fig. 4c) and 16% reduction in fat mass (Fig. 4i), compared to 46% (Fig. 1b) and 44% (Fig. 2b) reductions in whole-body knockouts. This discrepancy may stem from depot-specific differences in gene deletion efficiency. While Gpr146 was substantially depleted in iWAT, its reduction in eWAT diminished over time with HFD, despite efficient recombination in chow-fed young adults (Supplementary Fig. 7c, 7d). The reason for this apparent loss of knockdown efficiency in eWAT over time is not fully understood. One possible explanation is regional variation in Cre recombination efficiency under chronic metabolic stress. Additionally, obesity is known to induce substantial changes in adipose tissue composition where there is marked infiltration of immune cells and a corresponding reduction in the proportion of mature adipocytes33 - the specific cell type targeted by Adipoq-Cre. This deficit in mature adipocytes could dilute the overall depletion effect observed at the mRNA level when analyzing bulk tissue.

We next assessed systemic consequences of adipose Gpr146 deletion using indirect calorimetry. Unlike whole-body knockouts, adipose-specific knockouts did not exhibit increased energy expenditure despite the significant reduction in body weight and fat mass (Supplementary Fig. 7e, f). However, they displayed a significant increase in respiratory exchange ratio (RER) during the light cycle (Supplementary Fig. 7g), indicating a shift toward carbohydrate utilization.

Taken together, these findings demonstrate that adipose-resident GPR146, rather than hepatic GPR146, is primarily responsible for the regulation of diet induced obesity, hepatic steatosis and circulating FFA levels in response to HFD.

AAV-mediated acute knockdown of Gpr146 confirms its role in promoting hepatic steatosis and adiposity

To further validate the regulatory role of GPR146 in hepatic lipid accumulation and to rule out the possibility that the phenotypes observed in germline knockout mice result from developmental compensation, we employed an AAV-mediated approach to acutely knock down Gpr146 in adult mice. This strategy allows temporal control of gene silencing in fully developed animals, enabling us to assess the metabolic effects of GPR146 depletion in a mature physiological context. Following AAV-shRNA delivery, mice were fed a MASH-inducing diet rich in fat, cholesterol and fructose, which better recapitulates human liver inflammatory pathology34 (Fig. 5a). This approach achieved robust knockdown in the liver (~70% reduction in mRNA levels), along with significant depletion in both white and brown adipose tissues (Fig. 5b). Consistent with the findings from germline knockout models, AAV-mediated GPR146 depletion significantly reduced hepatic triglyceride content and suppressed the expression of key inflammatory markers in the liver (Fig. 5c, d).

Beyond the liver, AAV-mediated knockdown of Gpr146 in adult mice also led to significantly lower body weight and reduced white adipose depot mass (Fig. 5e-h). The most pronounced effect was observed in eWAT, which was reduced by approximately 40% compared to control littermates (Fig. 5g). These results reinforce the role of GPR146 in promoting diet-induced obesity and liver steatosis and confirm that its metabolic effects are not limited to developmental loss of function.

GPR146 Promotes Differentiation of both Mouse and Human Preadipocytes

In response to overnutrition, adipose tissue undergoes expansion through hypertrophy (enlarged adipocytes), hyperplasia (increased numbers of adipocytes) or both35. Given the observed reduced fat mass and adipocyte size in both adipose-specific and whole body Gpr146 knockout mice, we sought to investigate whether and how GPR146 regulates adipose biology.

We first isolated stromal vascular fraction (SVF) and adipocyte fraction from iWAT of Gpr146+/+ and Gpr146-/- mice fed with HFD for 3 months and assessed their gene expression profiles. In SVF fraction, we observed a significant reduction in the expression of Pparg and its downstream gene Fabp4, along with decreased expression of macrophage markers Adgre1 (F4/80) and Mcp1 in the absence of GPR146 (Fig. 6a). In adipocyte fraction, genes related to transport and uptake of fatty acid (Fabp4 and Cd36) were significantly suppressed upon GPR146 depletion, suggesting reduced lipid storage capacity within adipocyte (Fig. 6b). Consistent with our microarray data, we found a significant increase in the expression of Fasn, which encodes fatty acid synthetase, and Ppara, which encodes PPAR, the master transcriptional regulator for -oxidation, in adipocytes lacking GPR146 (Fig. 6b). Collectively, these results suggest that GPR146 might regulate both hypertrophic and hyperplastic expansion of adipose tissue.

To further study the regulatory role of GPR146 in adipogenesis, we characterized the expression pattern of GPR146 throughout the differentiation of mouse SVF and human preadipocyte SGBS cells. Notably, GPR146 expression gradually increased over time during differentiation, peaking in mature adipocyte (Supplementary Fig.9a, b). This observation aligns with the findings from adipose tissue single cell sequencing data showing that GPR146 is most highly expressed in mature adipocytes (Supplementary Fig.8b). In addition, the expression pattern of GPR146 closely resembled that of Pparg, suggesting a potential role for GPER146 in regulating adipogenesis (Supplementary Fig.9b). To test this hypothesis, we knocked down Gpr146 expression in mouse SVF using siRNA and found that the knockdown cells exhibited significantly lower total TG content compared to control cells following differentiation into adipocytes (Fig.6c, d and Supplementary Fig. 9Ac). To determine whether the reduced TG content reflected impaired commitment to adipogenesis, diminished cellular lipid accumulation, or both, we quantified the percentage of BODIPY-positive cells. GPR146 depletion significantly reduced the percentage of BODIPY-positive cells, indicating reduced adipocyte differentiation (Fig. 6e). Moreover, we employed a high-content imaging system to quantify the BODIPY-stained area of each differentiated adipocyte, serving as a proxy for TG content within individual adipocyte. Our findings indicate that the average BODIPY area of differentiated adipocytes from knockdown cells is smaller than control cells, suggesting a diminished TG accumulation in adipocytes upon GPR146 depletion (Fig. 6f, g). Gene expression analysis showed significantly lower levels of Pparg and Cebpa in Gpr146 knockdown cells compared to control cells following differentiation (Fig. 6h). while Cebpb, which encodes the transcription regulator typically activated in the early stage of differentiation and responsible for adipocyte commitment, was unaffected (Fig. 6h).

Therefore, these data suggest that GPR146 is more likely to regulate the later stages of adipocyte differentiation rather than the early commitment phase.

To validate these findings in a loss-of-function model, we performed ex vivo CRISPR/Cas9-mediated knockout of Gpr146 in SVF cells isolated from Cas9-expressing mice. Similar to the siRNA data, Gpr146 knockout significantly impaired adipocyte differentiation, as evidenced by reduced triglyceride accumulation (Supplementary Fig. 9d, e) and marked downregulation of Cebpa and Pparg (Supplementary Fig. 9f).

We next asked whether this regulatory role is conserved in human cells. Knockdown of GPR146 in SGBS cells also impaired adipocyte differentiation, as indicated by reduced lipid accumulation and lower expression of PPARG, CEBPA along with FASN and FABP4, which are involved in the terminal differentiation of adipocyte (Fig. 6i-l and Supplementary Fig. 9g). Additionally, we assessed mitotic clonal expansion (MCE), which has been shown to support early adipogenesis in both murine and human preadipocytes, by measuring using 5-bromodeoxyuridine (BrdU) incorporation 24 hours after induction of adipogenesis. GPR146 depletion significantly reduced BrdU incorporation in both SGBS and mouse SVF cells, suggesting that GPR146 regulates adipogenesis at least partially through MCE (Fig. 6m, n).

Interestingly, lentivirus-based overexpression of GPR146 in SGBS cells did not enhance adipogenesis. Instead, it significantly impaired differentiation, as shown by reduced lipid accumulation (Supplementary Fig. 9h, i). This data suggests that GPR146 does not operate through a simple dose-dependent mechanism. Rather, its expression must be maintained within an optimal range to preserve adipogenic capacity. Collectively, these gain- and loss-of-function studies in both mouse and human cells underscore the importance of finely tuned GPR146 activity in regulating adipocyte differentiation.

Altogether, these results establish that GPR146 promotes adipocyte differentiation in preadipocytes.

GPR146 Regulates Adipogenesis through Gαq-mediated Activation of PKC Signaling Pathway in both human and mouse preadipocytes

To elucidate the molecular mechanism by which GPR146 regulates adipogenesis, we performed phosphoproteomics profiling of SGBS cells with or without GPR146 knockdown following three days of differentiation. Pathway analysis by Metascape36 revealed an enrichment of phosphorylated proteins associated with cytoskeleton rearrangement, fatty acid synthesis and adipogenesis, suggesting a significant impact of GPR146 depletion on adipogenesis (Fig. 7a). Dysregulated adipogenesis can be influenced by one or more protein kinases and their downstream signaling cascades. To gain further insights into the potential kinases regulated by GPR146, we next conducted kinase substrate enrichment analysis (KSEA)37. This approach assesses changes in kinase activities resulting from GPR146 knockdown on the basis of previously annotated kinase substrates (PhosphositePlus) and predicted kinase-substrate relations (NetworKIN)37. KESA recapitulated the significant downregulation of multiple kinases involved in various pathways in GPR146 knockdown cells, including cAMP-PKA signaling (PRKAA2, PRKACG, PRKACA, PRKAC), mTOR signaling (RPS6KB1, RPS6KA1), PI3K-AKT signaling (AKT1, AKT2, PRKDC ), MAPK signaling (RAF1, MAPK8) and PKC signaling (PRKCG, PRKCA) (Fig. 7b).

To validate the findings from pathway and kinase substrate analysis, we examine the levels of PPARγ, C/EBP, phosphorylated AKT via western blot in GPR146 knockdown and control SGBS or mouse SVF cells. Knockdown of GPR146 resulted in decreased PPARγ and C/EBP levels and attenuated AKT phosphorylation in both human and mouse preadipocytes undergoing differentiation (Fig. 7c, Supplementary Fig. 10a-f), supporting impaired PI3K-AKT signaling. Given that insulin signaling activates PI3K-AKT to promote adipogenesis, in part by upregulating adipogenic transcription factors such as PPARγ38,39, our data suggest that reduced AKT activation underlies the impaired differentiation phenotype observed in GPR146-deficient cells.

Next, we proceed to investigate how GPR146 modulates AKT activation during adipogenesis. Upon ligand binding, G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins and facilitates the exchange of GTP for GDP. This process results in the dissociation of the G protein into its α and βγ subunits, which subsequently elicit a variety of downstream signaling pathways40. There are four major sub-families of Gα subunit (Gαs, Gαi/o, Gαq/11 and Gα12/13) that associate with GPCRs in a cell- and context-dependent manner, triggering different GPCR signaling depending on their subfamilies. Typically, Gαs stimulates the activation of adenyl cyclase and increases cAMP levels, which, in turn, leads to the activation of protein kinase A (PKA) and downstream effectors. Conversely, Gαi inhibits adenyl cyclase, resulting in lower cAMP levels41. To investigate whether GPR146 elicits its downstream signaling through Gαs/Gαi, we knocked down GPR146 in SGBS cells and mouse SVF cells followed by measuring the intracellular cAMP levels at different time points during differentiation. While we observed a trend of slightly increased intracellular cAMP levels in SGBS cells without GPR146, SVF cells upon GPR146 knockdown exhibited significantly decreased cAMP levels compared to control cells (Fig. 7d and Supplementary Fig. 10g). To examine if PKA signaling regulates adipogenesis, we used PKA inhibitor H89 to treat the preadipocytes during differentiation and found that it did not affect adipogenesis and AKT signaling activity in SVF cells (Supplementary Fig.10h-k). Therefore, these data collectively exclude the possibility of Gαs- or Gαi-mediated signaling pathways being responsible for attenuated adipogenesis in the absence of GPR146.

Given that PKC signaling pathway is also significantly downregulated in GPR146 knockdown cells from our phosphoproteomics study, we next tested whether GPR146 regulates adipogenesis by coupling to Gαq. Gαq upon activation binds to and activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol bisphosphate (PIP2) into two second messengers: diacylglycerol (DAG) and inositol triphosphate (IP3)41. DAG activates PKC, while IP triggers calcium release. We then measured cellular IP1 levels as a proxy for Gαq signaling. In SGBS cells, IP1 levels were comparable between GPR146 knockdown and control cells up to day 2 of differentiation. However, starting from day 3 of differentiation, knockdown cells exhibited a significant reduction in IP1, with a more pronounced decrease by day 4 (Fig. 7e). A similar reduction in IP1 was observed in SVF cells at day 3 after differentiation (Supplementary Fig.10l). Collectively, these data suggested that GPR146 likely regulates PKC signaling pathway through coupling to Gαq during adipogenesis.

To further interrogate if GPR146 regulates adipogenesis through Gαq-PKC axis, we treated SGBS and SVF cells with the PKC inhibitor GF109203X during differentiation. PKC inhibition significantly attenuated adipogenesis in both control (siNC) and GPR146 knockdown (siGPR146) cells compared to DMSO-treated counterparts. Notably, treatment with the PKC inhibitor eliminated the difference in adipogenic capacity between control and GPR146-deficient cells, suggesting that impaired PKC signaling underlies the differentiation defect caused by GPR146 loss in both human and mouse preadipocytes (Fig. 7f, g and Supplementary Fig. 10m-o). GF109203X also reduced phosphorylation of AKT and expression of PPARγ and C/EBPα in control (siControl) cells, with no further effect in knockdown(siGPR146) cells (Fig. 7h and Supplementary Fig.10p-r). Taken together, these data suggest that GPR146 promotes adipogenesis through Gαq-mediated activation of PKC and downstream AKT signaling.

GPR146 regulates mature adipocyte lipolysis through ERK signaling

Given the high expression of GPR146 in mature adipocytes, we next explored whether GPR146 influences adipocyte function beyond differentiation. Transcriptomic profiling of eWAT and iWAT from whole-body knockout mice revealed no significant changes in the expression of major adipokines, including Adipoq (adiponectin), Lep (leptin), Retn (resistin), and Cfd (adipsin) (Supplementary Fig. 11a, b), suggesting that GPR146 does not broadly impact endocrine output. Having established that GPR146 regulates adipogenesis through the Gαq-PKC-AKT signaling axis in preadipocytes, we next investigated whether this axis is still active in mature adipocytes. To test this, we first differentiate the SGBS cells into mature adipocytes, then knocked down GPR146 using siRNA (Fig. 8a and Supplementary Fig. 11c). Interestingly, unlike in preadipocytes, GPR146 knockdown in mature adipocytes did not alter the expression of PPARG and CEBPA, the key transcriptional regulators of adipogenesis (Supplementary Fig. 11c), nor did it affect insulin-stimulated AKT phosphorylation (p-AKT) (Supplementary Fig. 11d, e). These findings suggest that the GPR146-Gαq-PKC-AKT axis is primarily active during adipocyte differentiation, but not in mature adipocytes.

We then examined if GPR146 regulates lipolysis in mature adipocytes. Knockdown of GPR146 via siRNA significantly reduced forskolin-stimulated glycerol release, indicating impaired lipolytic activity (Fig. 8b). Conversely, doxycycline (DOX)-induced overexpression of GPR146 significantly enhanced glycerol release (Fig. 8c). Notably, GPR146 knockdown did not alter the mRNA levels of the three key lipases involved in lipolysis, LIPE (HSL), PNPLA2 (ATGL), and MGLL (MGL) (Supplementary Fig. 11c), suggesting that its regulation of lipolysis operates at the signaling level rather than through transcriptional control of these enzymes. Further mechanistic analysis revealed that ERK signaling activity was significantly decreased upon GPR146 knockdown and increased upon overexpression in mature adipocytes (Fig. 8d and Supplementary Fig. 11f-h). Importantly, pharmacological inhibition of ERK using PD98059 abolished the lipolysis-enhancing effect of GPR146 overexpression (Fig. 8c), supporting a model in which GPR146 promotes lipolysis through ERK-dependent mechanisms. To validate these findings in vivo, we assessed lipolysis in AAV-shERNA-mediated Gpr146 knockdown mice following stimulation with CL316,243, a 3-adrenergic receptor agonist. While CL316,243 induced modest lipolysis in control mice, Gpr146 knockdown significantly blunted glycerol release at 30 minutes post-treatment (Fig. 8e), consistent with our in vitro results.

In summary, these findings demonstrate that GPR146 regulates adipose function in a stage-specific manner-promoting adipogenesis via Gαq-PKC-AKT signaling during differentiation and stimulating lipolysis via ERK signaling in mature adipocytes-highlighting the context-dependent nature of GPR146-mediated metabolic control. In the absence of GPR146, impaired adipocyte differentiation and reduced lipolytic capacity together lead to diminished efflux of free fatty acids (FFAs) from adipose tissue. This reduction in FFA release limits hepatic FFA influx, thereby contributing to the observed protection against hepatic triglyceride accumulation and diet-induced fatty liver. These findings therefore position GPR146 as a key regulator of adipose-liver metabolic crosstalk under conditions of metabolic stress.

Methods
Mice and Diet

All animal care and experimental procedures conducted in this study received approval from the Institutional Animal Care and Use Committee of Harvard University and National University of Singapore. The mice were maintained in a controlled environment with a temperature of 23 ± 2°C and approximately 40% humidity, following a 12-hour light-dark cycle with free access to water and food unless otherwise stated. Male and female mice aged between 6 to 36-week-old were utilized in this study to ensure matching of age and sex. C57BL/6 mice were acquired from the Jackson Laboratory at 8 weeks of age. Gpr146 +/+ and Gpr146 -/- mice were generated through breeding of Gpr146 +/- mice. Adipose-specific and liver-specific GPR146 knockout mice were established based on our previous study by crossing Gpr146fl/fl mice with Adipoq-Cre mice or Albumin-Cre mice obtained from the Jackson Laboratory12. Additionally, Rosa26-Cas9 knockin mice were also purchased from the Jackson laboratory as well. For dietary intervention studies, 6-week-old mice were subjected to a high-fat diet (60/Fat, TD.06414, Envigo) for indicated period of time.

For AAV-mediated knockdown studies, 8-week-old male C57BL/6 mice received retro-orbital injections of adeno-associated virus serotype 8 (AAV8) carrying either a scramble control or Gpr146-targeting shRNA under the U6 promoter. One week post-injection, mice were placed on a MASH-inducing diet enriched in fat, cholesterol and fructose (TD160785, Envigo) for 13 weeks. At study endpoints, mice were euthanized by carbon dioxide (CO₂) inhalation followed by cervical dislocation, in accordance with institutional animal care and use guidelines.

Indirect Calorimetry and Metabolic Studies

Body composition was assessed using an EchoMRI-100 T system (Echo Medical Systems) in multiple cohorts of Gpr146-deficient mice, including whole-body knockout (Gpr146⁻/⁻) and control (Gpr146⁺/⁺) mice (both sexes) fed either chow or high-fat diet (HFD), as well as adipose-specific knockout mice subjected to HFD feeding. Indirect calorimetry and locomotor activity were continuously monitored using a comprehensive lab animal monitoring system (CLAMS; Columbus Instruments). Measurements were conducted after 2 weeks of HFD feeding in whole-body knockout mice and at later time points in tissue-specific knockout models. Energy expenditure was calculated based on oxygen consumption (VO₂), and body weights were recorded before and after the metabolic cage study.

Fasting glucose and fasting insulin levels were measured in an independent cohort that included both male and female Gpr146⁺/⁺ and Gpr146⁻/⁻ mice maintained on either standard chow or HFD for 2 months. Blood glucose was determined using a handheld glucometer (OneTouch Ultra Easy, LifeScan) following an overnight fast, and plasma insulin levels were quantified by ELISA according to the manufacturer's protocol (Abcam).

Plasma free fatty acid (FFA) concentrations were measured using the Free Fatty Acid Quantification Kit (Sigma-Aldrich) in separate cohorts of mice, including both sexes of whole-body Gpr146⁻/⁻ and Gpr146⁺/⁺ mice on HFD, as well as adipose-specific and liver-specific knockout models following HFD feeding. All assays were performed in accordance with the manufacturers’ instructions.

Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs)

For glucose tolerance tests, Gpr146 +/+ and Gpr146 -/- mice of both genders, which had been fed with chow or HFD for 2 months were individually housed and subjected to a 16 hour-fasting before the assay. After baseline blood glucose levels were measured using One Touch Ultra (LifeScan), glucose (1g/kg body weight) was injected through intraperitoneal route. Glucose levels were further determined at 15, 30, 60, 90, and 120 min post-injection. The insulin tolerance test was conducted after 4-hour fasting of the mice. After baseline blood glucose levels were measured, human insulin (0.75-1U/kg body weight) was injected, followed by monitoring the blood glucose levels at 15, 30, 60, 90, and 120 min post-injection.

Hepatic lipid content assay

Hepatic lipid extraction was perform as previously described with slight modifications44. In brief, snap-frozen liver samples were weighed and homogenized in ten volumes of ice-cold PBS. Thereafter, 200µl of the homogenate was transferred into 1,200 ul of chloroform: methanol (2:1; v/v) mixture, followed by vigorous vortex for 30 seconds. One-hundred microliters of ice-cold PBS was then added into the mixture and mixed vigorously for 15 seconds. The mixture was subsequently centrifuged at 1680 xg for 10 minutes at 4°C. Two-hundred microliters of the organic phase (bottom layer) was transferred into a new tube and evaporated for dryness. To dissolve the dried lipids, 200 µL of 1% Triton X-100 in ethanol was added, and the solution was continuously rotated for 2 hours. Triglyceride, cholesterol, and free fatty acid content were determined using the Infinity Triglycerides reagent (Thermo Scientific), Infinity Cholesterol reagent (Thermo Scientific), and the free fatty acid quantification assay kit (Abcam), respectively, following the manufacturer's instructions.

Isolation of the mouse SVF

Subcutaneous adipose tissue depots were dissected from male wildtype C57BL/6J or Rosa26-Cas9 knockin mice aged 8 to 11 weeks. The tissue was finely minced using scissors and then digested in KRPH buffer (20 mM HEPES ,5mM KH2PO4,1mM MgSO4.7H2),1mM CaCl2.2H2O,136mM NaCl,4.7mM KCL) supplemented with 1mg/ml collagenase A (Sigma-Aldrich) for 1 hour at 37 °C with gentle agitation. Following digestion, the tissue suspension was filtered through a 100 μm cell strainer and then centrifuged at 150 g for 10 minutes at room temperature. The floating adipocytes fraction was subsequently collected, while the pellet was resuspended in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptavidin (P/S) (Thermo Fisher Scientific) as stromal vascular fraction (SVF). The medium was replaced the following morning to remove any floating cells or debris.

Cell lines, Culture Conditions, Transfection and Infection

The Human Simpson–Golabi–Behmel syndrome (SGBS) cell line was kindly supplied by Martin Wabitsch (University of Ulm, Germany)45 and authenticated as preadipocytes based on their response to adipogenic cues (cocktails) and the expression of adipogenic marker genes. The SGBS cell line was originally derived from human adipose tissue obtained with informed consent and with approval from the Ethical Committee of the University of Ulm, as described previously45. No additional ethical approval was required for its use in the present study. The SGBS cells were cultured in a basal medium of DMEM/F12 medium supplemented with 10% FBS, 3.3mM biotin,1.7 mM pantothenic acid and 100 U/ml penicillin-streptomycin (Sigma-Aldrich). To knock down the expression of GPR146 in human SGBS or mouse SVF cells, RNAimax (Thermo Fisher Scientific) reagent was used following the manufacturer’s instructions. To knock out Gpr146 in mouse SVF cells isolated from Rosa26-Cas9 mice, lentivirus expressing sgRNAs targeting GPR146 were incubated with SVF cells for 16h, after which the medium was replaced. For overexpression studies, SGBS cells were transduced with doxycycline-inducible lentiviral vectors expressing GFP or GPR146 and treated with 1 μg/mL doxycycline during the experimental period as indicated.

Adipocyte differentiation

Adipogenic differentiation of SGBS cell was induced by treating confluent cells with serum-free quick-Diff medium comprising 0.01mg/ml transferrin, 20 nM insulin, 100 nM cortisol, 0.2 nM tri-iodothyronine (T3), 25 nM dexamethasone, 250 µM isobutyl-1-methylxanthine (IBMX) and 2 μM rosiglitazone (Sigma-Aldrich). After 4 days, the medium was replaced with DMEM/F12 containing 3.3 mM biotin,1.7 mM pantothenic acid, 0.01 mg/ml transferrin, 20 nM insulin, 100 nM cortisol, 0.2 nM tri-iodothyronine every other day (Sigma-Aldrich).

For differentiation of mouse SVF cells, the cells were first cultured to confluence in DMEM/F12 medium supplemented with 10% FBS and 1% P/S, induced to differentiation in the adipogenic cocktail consisting 1μM dexamethasone, 5 μg/ml Insulin, 0.25 mM IBMX, 5 μM rosiglitazone, followed by maintenance treatment with 1 μg/ml Insulin and 5 µM Rosiglitazone every 48h until fully differentiated. Eight to ten days after differentiation, the differentiated adipocytes were stained with BODIPY (Sigma-Aldrich) or collected for RNA and protein extraction.

Quantification of in vitro differentiation

Oil-red O staining

For Oil-red O staining, differentiated adipocytes were rinsed with PBS twice and then fixed in 4% (v/v) paraformaldehyde solution for 20 min. Thereafter, the fixed cells were washed twice with PBS, followed by a 5 min incubation with 60 % isopropanol. The cells were then stained with Oil-Red O isopropanol solution for 15 min at room temperature, washed with PBS and imaged.

BODIPY staining

To assess differentiation potential, fully differentiated adipocytes were stained with 1 μM BODIPY for 20 minutes, followed by washing with PBS and imaged using the PerkinElmer Operetta high-content imaging system. The percentage of BODIPY-positive cells and the areas of the BODIPY stained adipocyte were quantified using Image J.

Histological analyses

Following dissection, epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT) and liver were promptly fixed in a 10% formalin-buffered solution (Sigma-Aldrich). Next, the fixed tissues were embedded, sectioned, and stained with haematoxylin and eosin (H&E) (Sigma-Aldrich). Histopathological images were captured with a digital pathology scanner (Aperio Versa 200, LEICA) and analysed with Image J (National Institutes of Health) and Adipo soft46 software to measure the area of adipocytes. The areas of 150 – 300 cells from two sections from each sample were measured.

In vitro and In vivo lipolysis

To assess the role of GPR146 in lipolysis, both in vitro and in vivo assays were performed. For in vitro analysis, differentiated human SGBS adipocytes were subjected to GPR146 knockdown using siRNA (ThermoFisher) or overexpression via doxycycline-inducible lenti-viral vectors expressing either GPP (control) or GPR146. Glycerol release was measured under basal and forskolin-stimulated conditions using a colorimetric assay kit (Sigma-Aldrich).

For in vivo lipolysis, male mice with AAV-mediated Gpr146 knockdown or scramble control, following 6 weeks of MASH diet feeding, were fasted for 4 hours and injected intraperitoneally with β3-adrenergic agonist CL-316,243 (1 mg/kg body weight) (MCE). Plasma glycerol levels were quantified at baseline, 30-, 60-, and 120-minutes post-injection.

cAMP/Gs and Gαq- IP1assay

To investigate the signalling downstream of GPR146, cellular cAMP level, which reflects the activation status of Gαs or Gαi signalling, was quantified using cAMP-Gs dynamic kit (PerkinElmer) following the manufacture’s protocol. Human SGBS cells or mouse SVF cells were transfected with siRNA to knock down the expression of GPR146 and then induced to differentiation with adipogenic cocktail. Cells were collected on day 0,1,2,3 and 4 after differentiation, resuspended in PBS and used for the cAMP-Gs assay. Specifically, 7 ul of the cell suspension was incubated with HTRF reagents (kit components) for 1 hour at room temperature, followed by the measurement of the fluorescence ratio at 665/620nm. Data were reported as the mean ± standard error of the mean (SEM) of three replicates.

Similarly, the activation status of Gαq signalling was probed following GPR146 knockdown by performing an IP1 assay using the IP-One Gq Kit (PerkinElmer). Transfected human SGBS preadipocytes and mouse SVF undergoing differentiation were harvested at indicated time points, and the endogenous IP1 level was quantified by measuring HTRF signal at 665/620 nm. Collectively, these data suggested that GPR146 likely regulates PKC signaling pathway through coupling to Gαq during adipogenesis.

Seahorse XF mitochondrial stress test

SGBS preadipocytes were differentiated into mature adipocytes as previously described. Fully differentiated cells were transfected with control or GPR146 siRNA, and 48 h after transfection, mitochondrial respiration was assessed using the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, Santa Clara, CA, USA; Cat. No. 103015-100) according to the manufacturer’s instructions. Oxygen consumption rate (OCR) was measured using a Seahorse XFe24 Analyzer (Agilent Technologies) under basal conditions and following sequential injection of oligomycin, FCCP, and rotenone/antimycin A.

Metabolomics

Mouse liver samples (average weight of 53.5 mg) were homogenized in four volumes of water per milligram of tissue (4 µL/mg) using a TissueLyser II bead mill (QIAGEN; Germantown, MD), with two 3 mm tungsten carbide beads and two 120-second intervals of high-speed shaking at 20 Hz. Homogenates were then aliquoted for analyses of lipids and polar metabolites using four complementary liquid chromatography tandem mass spectrometry (LC-MS) procedures as described previously47.

HILIC–positive mode (HILIC-pos)

Water-soluble metabolites were analyzed using a Shimadzu Nexera X2 UHPLC coupled to a Q Exactive Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific). Liver homogenates (10 µL) were protein-precipitated with nine volumes of acetonitrile/methanol/formic acid (74.9:24.9:0.2, v/v/v) containing stable isotope-labeled internal standards (valine-d8 and phenylalanine-d8). After centrifugation (9,000 × g, 10 min, 4°C), supernatants were injected onto an Atlantis HILIC column (150 × 2 mm, 3µm; Waters). Elution was performed at 250 µL/min starting with an isocratic hold at 5% mobile phase A (10 mM ammonium formate, 0.1% formic acid) for 0.5 min, followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 min. MS data were acquired in positive electrospray mode (m/z 70–800) at 70,000 resolution. Source settings were: sheath gas 40, sweep gas 2, spray voltage 3.5 kV, capillary temperature 350°C, heater temperature 300°C, S-lens RF 40, microscans 1, AGC target 1e6, and maximum ion time 250 ms.

HILIC–negative mode (HILIC-neg)

Targeted analysis of polar metabolites was performed on an ACQUITY UPLC system (Waters) coupled to a 5500 QTRAP mass spectrometer (SCIEX). Liver homogenate (30 µL) was extracted with four volumes of 80% methanol containing isotope-labeled internal standards (inosine-15N4, thymine-d4 and glycocholate-d4). Following centrifugation (9,000 × g, 10 min, 4 °C), supernatants were analyzed using a Luna NH₂ column (150 × 2.0 mm; Phenomenex) at a flow rate of 400 µL/min. The gradient began with 10% mobile phase A (20 mM ammonium acetate, 20 mM ammonium hydroxide in water) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 acetonitrile/methanol), ramping linearly to 100% A over 10 min. Detection was carried out in negative electrospray mode using multiple reaction monitoring, with compound-specific declustering potentials and collision energies optimized using authentic standards. The ion spray voltage was -4.5 kV and the source temperature was 500°C.

Lipid profiling (C8-positive)

For global lipid profiling, liver homogenates (10 µL) were mixed with 190 µL of isopropanol containing 1,2-didodecanoyl-sn-glycero-3-phosphocholine (Avanti Pola Lipids; Alabaster, AL) as an internal standard to precipitate proteins and extract lipids. Following centrifugation, the supernatants were injected directly onto a ACQUITY BEH C8 column (100 × 2.1 mm, 1.7 µm; Waters). The mobile phases were (A) 95:5:0.1 (v/v/v) 10 mM ammonium acetate/methanol/formic acid and (B) 99.9:0.1 (v/v) methanol/formic acid. The elution gradient began with an isocratic hold at 80% A for 1 min, followed by a linear ramp to 80% B over 2 min, a linear increase to 100% B over 7 min, and a final isocratic hold at 100% B for 3 min. MS data were acquired in positive electrospray ionization (ESI) mode using full-scan analysis over 200–1000 m/z at 70,000 resolution and 3 Hz data acquisition rate. Source parameters were set as follows: spray voltage 3 kV, capillary temperature 300°C, heater temperature 300°C, sheath gas 50, sweep gas 5, S-lens RF 60, in-source CID 5 eV, microscans 1, automatic gain control target 1e6, and maximum ion time 100 ms. Lipid species were identified by comparison with reference plasma extracts and annotated based on the total acyl chain carbon count and double bond content.

Reversed-phase analysis (C18–negative)

Analyses of free fatty acids, bile acids, and metabolites of intermediate polarity were conducted using a Shimadzu Nexera X2 U-HPLC coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific). Liver homogenates (30 µL) were extracted using 90 µL of methanol containing PGE2-d4 as an internal standard (Cayman Chemical Co.; Ann Arbor, MI) and centrifuged (10 min, 9,000 × g, 4°C). The supernatants (10 µL) were separated on an ACQUITY BEH C18 column (150 × 2.1 mm; Waters) at a flow rate of 450 µL/min. The mobile phases consisted of (A) 0.01% formic acid in water and (B) 0.01% acetic acid in acetonitrile. The gradient started isocratically with 20% A for 3 minutes, followed by a linear gradient to 100% mobile phase B over 12 minutes. MS detection was performed in negative ESI mode (m/z 70-850) at a resolution of 70,000 (3 Hz). Additional MS settings included: spray voltage, -3.5 kV; capillary temperature, 320°C; probe heater temperature, 300 °C; sheath gas, 45; auxiliary gas, 10; and S-lens RF level 60.

Data Processing

Raw data files from the Exactive Plus and Q Exactive instruments were processed using TraceFinder (Thermo Fisher Scientific) and Progenesis QI (Nonlinear Dynamics). Data acquired on the 5500 QTRAP were processed using MultiQuant (SCIEX). For each method, metabolite identities were confirmed using authentic reference standards or reference samples.

Immunoblotting

For immunoblotting analyses, cells or tissues were lysed in RIPA buffer containing the protease and phosphatase inhibitor cocktail, and the protein concentration was estimated using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein were subjected to SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, followed by incubations with primary and secondary antibodies.

Gene expression analysis

Total RNA was extracted from cells or tissues using Trizol (Thermo Fisher Scientific) or the RNeasy kit (QIAGEN). 1 µg of RNA was reverse transcribed using High Capacity cDNA Reverse transcription kit (Thermo Fisher Scientific). Quantitative real-time PCR(RT-PCR) was performed on La ViiATM 7 RT-PCR system (Applied Biosystems Inc.) with SYBR-green ChamQTM SYBR Color qPCR Master Mix (Vazyme). Relative expression level for gene of interest was compared to the housekeeping gene RPLP. Primer sequences used in this study was listed in supplementary table 2.

Genome-wide mRNA expression profiles in mouse eWAT, iWAT, BAT, and liver were assessed with the Affymetrix GeneChipTM Mouse Gene 2.1 ST Array Strip in accordance with the manufacturer’s instructions. Gene set enrichment analysis (GSEA) was performed, and significantly enriched terms were identified using a false discovery rate q value of less than 0.05.

Phosphoproteomics

Sample preparation

Three replicates of control and GPR146 knock down SGBS cells were harvested by scraping into lysis buffer (8 M urea, 50 mM Tris-HCl, pH 8.0 with protease and phosphatase inhibitor cocktails) directly after two PBS washes. The cell pellets were sonicated for 3 cycles and the protein concentration was determined using Pierce BCA Protein Assay Kit following the manufacturer’s instructions. All samples (approximately 200 µg each) were subjected to reduction with 20 mM tris (2-carboxyethyl) phosphine (TCEP) in 100 mM triethylammonium bicarbonate buffer (TEAB) and alkylated by 55 mM chloroacetamide (CAA) at room temperature for 30 min in the dark. The urea concentration was then diluted to 1 M using 100 mM TEAB, pH 8.5 for digestion. The samples were digested with 5 µg of Lys C for 4 h, followed by overnight digestion with 5 µg of modified trypsin at 25 °C. The efficiency of digestion was evaluated and considered acceptable if protein missed cleavage was 10% or lower. The resulting peptides were acidified and desalted with a 60 mg HLB cartridge (Waters) as per manufacturer’s instructions. Briefly, the cartridge was first activated with 0.5 ml of 100% acetonitrile and then equilibrated with 1 ml of 0.5% acetic acid in water, followed by peptide sample loading. Bound peptides were washed twice with 2 mL of 0.5% acetic acid in water and eluted with 0.5 mL of 0.5% acetic acid in 65% acetonitrile.

Approximately one-tenth of the desalted samples was retained as a proteome input control, while the remaining samples were used for phosphorylated peptide enrichment.

Phosphopeptides were enriched from the desalted peptides with TiO beads. The samples were resuspended in a final composition of 6% trifluoroacetic acid (TFA) in 40% acetonitrile and incubated with the TiO beads at 25 °C for 10 min. This incubation step was repeated once more with fresh beads. Beads from both enrichments were combined, and a sequential wash was performed with 6% TFA in 10%, 40%, and 60% acetonitrile. The elusion was carried out first with 5% ammonia water first followed by 5% ammonia water in 25% acetonitrile.

Both the input and phosphopeptides samples were dried using vacuum centrifugation, resuspended with 100 mM TEAB (pH 8.5) and labelled individually for 2 hours with TMTsixplex™ Isobaric Label Reagent (Thermo Fisher Scientific). After quenching with 1 M ammonium formate (pH 10), the labelled samples were pooled together according to their respective proteomics input set and phosphopeptide set. Subsequently, the samples were desalted and eluted with 10 mM ammonium formate (pH 10) in increasing acetonitrile concentration of 14%, 18%, 21%, 24%, 27%, 32% and 60%, resulting a total of 7 fractions for each sample set. Following two acidified washes, the fractions were ready for data acquisition.

Mass spectrometry

Both the proteomics input samples and phosphopeptides samples were subjected to mass spectrometry analysis using reverse phase liquid chromatography on the nLC1000 UHPLC system connected to Thermo Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Each fraction was separated on a 50 cm length x 75 µm internal diameter, 2 µm particle size Easy Spray column (Thermo Fisher Scientific) over a 70-min gradient with pre-programmed mixing of solvent A (0.1% formic acid in water) and solvent B (99% acetonitrile, 0.1% formic acid in water). The following acquisition parameters were applied for all samples: data dependent acquisition (DDA) in positive ion mode with survey scan of 60,000; scan range of 350-1550 m/z, and AGC target of 4e5; MS/MS analysis using Orbitrap mass analyzer at higher energy collision dissociation of 42%; AGC target of 7.5e4; isolation window of 1 m/z; and maximum injection time of 100 ms.

Quantification and Statistical Analyses

The statistical significance of differences was determined by unpaired, two-tailed Student’s t test (two groups) or oneway ANOVA (more than two groups), corrected p values < 0.05 considered statistically significant. Asterisks denote corresponding statistical significance *p < 0.05 and **p < 0.01. For mouse liver western blotting, each sample corresponds to protein extract from one mouse.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present findings that reveal a role for GPR146 in the prevention of hepatic steatosis. They propose that it is GPR146's role in adipose tissue and lipid metabolism that prevents TG accumulation in the liver. GPCRs represent attractive drug targets and GPR146 has intriguing biology, however, several key questions remain.\nIf GPR146 KO affects adipogenesis, is that really desirable? The authors show similarities in pattern to the master adipose regulator Pparg and yet knocking out Pparg/disrupting adipogenesis is deleterious on metabolic health, including the liver. Especially since knocking down GPR146 affects hyperplasia (which is the healthier expansion compared to hypertrophy). If the adipose tissue isn’t expanding and the mice eat the same amount of HFD, as shown by the authors, then where is the extra fat going? Finally, if GPR146 affects adipogenesis, why don’t they observe lipodystrophy in the whole body or adipose specific KO?\nRegarding the energy expenditure, several questions remain. Is the increase in catabolism that was observed in vivo also present in isolated adipocytes? What is the in vivo energy expenditure profile in the adipose specific KOs? Does the increased energy expenditure persist at thermoneutrality? How about on chow? The authors say the mechanism is not through canonical thermogenic cycles but Adrb3, which they show is up, is the major driver of both Ucp1 and non-Ucp1 dependent thermogenesis. And CL-316,243 does far more than just stimulate lipolysis.\nWhy do the author’s think that knocking Gpr146 down in cells has an effect when they haven’t added any ligand? Is it known if the ligand for GPR146 is endogenously produced?\nAll the in vivo findings involve prevention of steatosis because of constitutively expressed genetic models. Inducibly depleting Gpr146 after NAFLD has developed would significantly strengthen the manuscript.\nMinor\n-In the discussion, just because Ucp1 expression is unchanged does not mean the EE is Ucp1 independent.", "reviewer_2": "In this manuscript, the authors investigated the regulatory role of the G protein-coupled receptor 146 (GPR146) in adipose tissue and liver lipid metabolism. In an in vivo model, GPR146 deficiency exhibited a protective effect against obesity induced by a high-fat diet, reducing liver steatosis and accompanying a decrease in circulating liver enzyme levels. Global depletion of GPR146 in mice affected energy homeostasis by increasing energy expenditure during high-fat diet feeding. The study revealed that specific depletion of GPR146 in adipocytes in mice could counteract diet-induced fatty liver by limiting the influx of free fatty acids. Additionally, the authors demonstrated that GPR146 deletion in vitro inhibits adipogenesis by blocking the activation of Gαq-PKC AKT signaling.\nThe subject matter of this study holds considerable significance in the realm of lipid metabolism. Nevertheless, the data outlined in the current version do not entirely substantiate the authors' conclusions. A notable deficiency lies in the absence of a mechanistic explanation detailing how GPR146 influences lipid turnover and adipogenesis. Here are specific comments aimed at potentially enhancing the manuscript.\n1. The first question inquires whether the protein expression level of GPR146 is elevated in the adipose tissue of obese individuals compared to non-obese ones.\n2. Does the expression level of GPR146 in wild-type mice also increase over time in various adipose tissues (eWAT, ingWAT, and BAT) during HFD feeding?\n3. Global depletion of GPR146 in mice, as observed in Fig 2b by MRI measurements, led to an increase in the percentage of lean mass under HFD feeding. However, the data only indicate the percentage of lean mass. Is there an increase in the absolute amount of lean mass as well? Utilizing a whole-body composition analyzer might offer more precise in vivo measurements the mass of body lean, fat, and fluid.\n4. Does the protective role of GPR146 deficiency against high-fat diet (HFD)-induced obesity suggest a contrasting effect when GPR146 is up-regulated? The authors should provide or at least discuss the results induced by GPR146 over-expressions.\n5. Moreover, the data from GPR146 deficiency do not entirely support the authors' conclusions regarding the promoting effects of GPR146 on adipogenesis. To strengthen this assertion, the author should include data from GPR146 overexpression experiments.\n6. The author discussed ATP-consuming futile cycles in whole-body GPR146 deficiency. It raises the question whether similar findings could be observed in mice with adipocyte- or hepatocyte-specific GPR146 deficiency.\n7. Since PKC is the endpoint of the proposed GPR146 signaling pathway, in the GPR146 depletion background, inhibition of PKC function by pharmacological reagent alone cannot support the authors' hypothesis that Gαq-PKC-pAkt is downstream of GPR146 signaling.\n8. To address the roles of GPR146 in lipid turnover, the authors should provide more functional experiments, instead of just differential expressions analyses.\nMinor points:\n1. It would be helpful to provide a brief description of G protein-coupled receptors in mediating functional activities of adipose tissues.\n2. The image should include a scale bar for reference.\n3. Please include the sequences for the siRNA used.", "reviewer_3": "The study by Shi et al. provides a large set of data investigating the effects of GPR146 on liver steatosis and particularly the role of adipocyte GPR146. The authors first describe findings extracted from public GWAS databases indicating association of GPR146 variants with increased plasma liver enzymes and CRP. In the following experiments they show that GPR146 knockout reduces liver steatosis and plasma ALT in mice. Liver metabolomics was used to characterize in more detail effects on lipids. GPR146 knockout also decreased weight gain and fat mass in response to HFD that appears to be related to elevated energy expenditure during resting periods. The authors used tissue specific knockout models to demonstrate that Adipoq-Cre but not Alb-Cre induced knockout alleviated the liver steatosis suggesting that adipocyte GPR146 mediates the steatotic effect. Adipocyte KO of GPR146 also reduced weight gain, fat mass and plasma FFA. In cell models GRP146 was found to promote adipogenesis potentially through Gαq-PCK-AKT pathway. The study is interesting and thorough. However, there are several points of concern:\n1. The effect of Adipoq-Cre induced knockout on Grp146 expression in WAT requires further information. At least after 4 months of HFD the effect of the KO on Gpr146 expression appears either limited (ingWAT) or not existing (eWAT) (Extended Figure 7e). Please provide necessary evidence of the effect of Grp146 KO on GPR146 expression in different time points used in the study. The current data raises questions about the mechanisms of the observed effects in Adipoq-KO mice.\n2. The authors state in several instances that GRP146 KO ameliorates hepatic inflammation. However, I don’t think any evidence is presented to support this notion. ALT cannot be considered and an inflammation marker. However, indeed it would be necessary to study the role of GPR146 in relation to liver inflammation and fibrosis. Simple HFD model may not be the best for this purpose, but it may require additional factors such as fructose feeding. The authors also reported elevated CRP associated with GPR146 variants in humans. What about in mice? Does the Gpr146 KO affect CRP?\n3. The effect on glucose metabolism should be better explained. Do the increased liver hexose metabolites indicate upregulation of hepatic glucose uptake or what is the authors’ interpretation? It is not clear what the authors mean by statement that “GRP146 deficiency promotes glucose mobilization”, page 5 line 9-10. Indeed, glycogen storage was increased.\n4. The authors claim that GRP146 KO ameliorates insulin resistance. However, this is not obvious. The KO improves glucose tolerance (fig. 2g) which could suggest better insulin tolerance. However, in ITT the effects can be seen only in late time points when the animals are recovering from the insulin effect. A true difference in insulin sensitivity should be seen in early time points when glucose is decreasing. This is especially obvious in female mice where the effect in ITT is observed only after 90 min (Ex. Fig. 3d) but also to some extent in male mice (Fig 2h). The results rather appear to suggest defects in counter-regulatory mechanisms correcting insulin-induced hypoglycemia. Also, there was no difference in plasma fasting insulin (Ex. Fig. 3e) again suggesting no clear effect on insulin sensitivity. HOMA-IR index, preferably calculated with the mouse modified formula (doi: 10.1177/0023677212473714) could be provided also.\n5. The authors provide transcriptomics data suggesting decreased macrophage infiltration in the adipose deposits of the GRP146 KO mice. Is this supported by any other data such as histology?\n6. The study includes several types of omics data; however, the full data has not been made available and no repository for data storage has been indicated?"}, "author_response": "Point-by-Point Response\nWe sincerely thank the reviewers for all insightful comments. In response, we have\nthoroughly revised the manuscript and addressed all reviewer comments with substantial new\ndata and clarifications.\nWe sincerely apologize for the substantial delay in submitting this revision and greatly\nappreciate the patience and understanding of the reviewers and editorial team. After my\ntransition to an independent faculty position, we encountered an unexpected issue:\nthe original Gpr146 whole-body knockout mouse strains had incorrect genotypes due to\ncolony maintenance by a collaborator after the closure of my former postdoctoral lab. We\ntherefore reconstructed and revalidated this line, which required considerable time and effort\nbut was essential to ensure experimental rigor and reproducibility.\nDuring this period, we have substantially expanded our investigation and uncovered new\nmechanistic insights, including the distinct roles of GPR146 in regulating adipogenesis and\nlipolysis via the Gαq-PKC-AKT and ERK signaling pathways, respectively. These findings\nfurther clarify how GPR146 modulates adipose-liver crosstalk and contributes to hepatic\nsteatosis. We have also included new in vivo experiments using AAV-mediated knockdown\nwith NASH diets to demonstrate the translational relevance of targeting GPR146 in adult\nanimals.\nIn parallel, we have taken great care to thoroughly restructure the manuscript to\nenhance clarity, logical flow, and scientific rigor. Throughout, we have also tempered our\nlanguage to avoid overinterpretation and ensure our conclusions are appropriately supported\nby the data. We hope this revised version fully addresses the concerns raised and is now\nsuitable for further consideration.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors present findings that reveal a role for GPR146 in the prevention of hepatic\nsteatosis. They propose that it is GPR146's role in adipose tissue and lipid metabolism that\nhas intriguing biology, however, several key questions remain.\n1. If GPR146 KO affects adipogenesis, is that really desirable? The authors show similarities\nin pattern to the master adipose regulator Pparg and yet knocking out Pparg/disrupting\nadipogenesis is deleterious on metabolic health, including the liver. Especially since\nknocking down GPR146 affects hyperplasia (which is the healthier expansion compared to\nhypertrophy). If the adipose tissue isn’t expanding and the mice eat the same amount of\nHFD, as shown by the authors, then where is the extra fat going? Finally, if GPR146 affects\nadipogenesis, why don’t they observe lipodystrophy in the whole body or adipose specific\nKO?\nAnswer: We thank the reviewer for this insightful and important question. We fully agree that\nexcessive inhibition of adipogenesis can be deleterious to metabolic health, as highlighted in\nstudies of PPARG deficiency. However, our data suggest that GPR146 does not serve as a\nbinary on/off switch for adipogenesis but rather functions as a modulator of this process. And\nimportantly, the reduced lipid storage capacity observed in GPR146-deficient mice is\naccompanied by increased energy expenditure, which likely represents the primary driver of\nthe overall lean phenotype and diminished hepatic lipid accumulation.\nSpecifically, GPR146 deficiency does not abolish adipogenesis but leads to a partial\nattenuation of terminal differentiation, as evidenced by a selective reduction in mid-to-late\nstage transcription factors (PPARγ and C/EBPα), while early commitment factors such as\nC/EBPβ remain unchanged (Fig. 6h, l). This suggests that GPR146 regulates adipocyte\nmaturation rather than commitment. This allows for the formation of histologically intact, but\nsmaller adipose depots, and importantly, we do not observe features of lipodystrophy in either\nthe global (Fig. 2c, d, g, h) or adipose-specific knockout mice (Fig. 4k, l).\nAs GPR146 is most highly expressed in mature adipocytes, we further investigated its role in\nregulating lipolysis. In this revision, we present new data showing that doxycycline-induced\noverexpression of GPR146 in SGBS-derived adipocytes significantly enhances lipolysis (new\ndata, Fig. 8c) while both siRNA-mediated knockdown of GPR146 in SGBS adipocytes (new\ndata, Fig. 8b) and acute depletion of GPR146 in mice using AAV-shRNA significantly reduces\nlipolytic activity (new data, Fig. 8e). These findings are consistent with the reduction in\ncirculating free fatty acid (FFA) levels observed in both global (Fig.1k) and adipose-specific\nGPR146 knockout mice (Fig. 4e, f), which likely contribute to the reduced FFA influx into the\nliver and subsequent attenuation of hepatic TG accumulation.\nAt the whole-body level, in terms of energy balance, GPR146-deficient mice exhibit a\nsignificant increase in resting energy expenditure, particularly during the light (inactive)\nphotoperiod in males (Fig. 3d). Honestly, while we present robust data demonstrating that\nadipose-specific depletion of GPR146 attenuates both adipogenesis and lipolysis-thereby\nreducing FFA efflux and contributing to protection against hepatic steatosis- the precise\nmolecular mechanism by which GPR146 deficiency enhances energy expenditure remains\nunclear in male mice. Although we initially speculated the involvement of ATP-consuming futile\ncycles, our evidence is limited to mRNA expression data. Nevertheless, this elevated energy\nexpenditure likely play a key role in offsetting the reduced capacity for adipose expansion,\nthereby preventing ectopic lipid accumulation.\nIn our revised study, we also performed indirect calorimetry in female GPR146-deficient mice\nand found that KO females exhibited significantly increased energy expenditure compared to\ncontrols during both light and dark cycles (new data, Fig. 3h, i). Interestingly, in contrast to\nmale KO mice where Ucp1 expression remained unchanged in eWAT and ingWAT\n(Supplementary Fig. 6f, g), female KO mice displayed a more than 15-fold increase in Ucp1\nexpression specifically in eWAT, along with marked upregulation of Pgc1α and Ppara (new\ndata, Supplementary Fig. 6h, i). These data suggest that in females, activation of UCP1-\nmediated thermogenesis likely contribute to the observed increase in energy expenditure,\npotentially through enhanced mitochondrial biogenesis and oxidative metabolism. This\nemerging sex difference in thermogenic activation may reflect differential hormonal regulation\nor adipose tissue plasticity between males and females1,2, and further investigation to\nelucidate sex-specific mechanisms underlying GPR146-dependent metabolic control is\nrequired.\nImportantly, we would like to highlight the systemic metabolic reprogramming in GPR146-\ndeficient mice. The knockout mice exhibit improved glucose handling, accompanied\nby increased hepatic glycogen content and elevated levels of hexose and pentose phosphate\nintermediates, suggesting a possible enhancement in hepatic insulin sensitivity (Fig. 2k, l and\nSupplementary data Fig. 3a, b, c). The accumulation of pentose phosphate intermediates\nfurther suggests increased NADPH production3, which could contribute to redox\nhomeostasis and protection from oxidative stress, a key driver of hepatic inflammation in\nNAFLD4.\nAdditionally, untargeted metabolomics also revealed significantly elevated hepatic levels of\nseven standard amino acids in Gpr146-/- mice, namely histidine, cysteine, lysine, valine,\nleucine, isoleucine, and phenylalanine (new data, Supplementary Fig. 3d). Consistent with\nthese hepatic metabolic changes, chow-fed Gpr146-/- mice exhibited a significantly higher\nrespiratory exchange ratio (RER) during the dark cycle compared to controls (new data,\nSupplementary Fig. 3e, f), indicating a greater reliance on glucose as an energy source.\nFurthermore, chow-fed Gpr146-/- mice displayed significantly increased lean mass without\nchanges in fat mass (new data, Supplementary Fig. 3g, h), potentially reflecting altered\nsystemic nutrient partitioning and increased availability of amino acids at chow-fed baseline\ncondition. Together, these findings support a model in which GPR146 deficiency induces\ncoordinated metabolic shifts that reduce the need for lipid storage and limit ectopic lipid\naccumulation in the liver.\nWe have revised the manuscript to reflect this updated mechanistic interpretation and thank\nthe reviewer for prompting a deeper and more critical evaluation of these data.\nFigure 8. GPR146 regulates mature adipocyte lipolysis through ERK signaling. b,\nGlycerol release from SGBS mature adipocytes transfected with siGPR146 or control siRNA,\nwith or without forskolin stimulation; values normalized to intracellular triglyceride (n=3\nreplicates per experiment, representative of 3 independent experiments, by Student’s t test).\nc, Glycerol release from SGBS adipocytes with doxycycline-inducible GPR146.\nMeasurements were performed with or without the MEK inhibitor PD98059 and normalized to\nintracellular TG (n=3 replicates per experiment, representative of 3 independent experiments,\nby Student’s t test). e, In vivo lipolysis assay in mice with acute depletion of Gpr146 using\nAAV-shRNA or scramble control, maintained on a MASH-inducing diet for 12 weeks. Mice\nwere injected with CL-316,243 to stimulate lipolysis, and plasma glycerol levels were\nmeasured at the indicated time points. (n=7-9 mice per group, by Student’s t test). * p< 0.05,\n** p< 0.01; bars indicate mean± s.e.m..\nFigure 3. GPR146-deficient Mice exhibit Elevated Energy expenditure. Indirect\ncalorimetry study in female mice fed HFD for two weeks. h,i, Total energy expenditure (EE)\nduring the light (h) and dark (i) phases, averaged over four consecutive days, expressed as\nkcal/kg lean mass. * p< 0.05, ** p< 0.01; bars indicate mean± s.e.m..\nSupplementary Figure 6, Metabolic phenotyping and thermogenic pathway regulation\nin adipose tissue of GPR146-deficient and control mice, Related to Fig3. h,i, qPCR\nexpression analysis of Ucp1, Pgc1a and Ppara in eWAT (h) and iWAT (i) from female\nGpr146+/+ and Gpr146-/- littermates fed HFD (n=4-8 mice per group, by Student’s t test). * p<\n0.05, ** p< 0.01; bars indicate mean ± s.e.m..\nSupplementary Figure 3. GPR146 deficiency reprograms hepatic glucose and amino\nacid metabolism. d, Relative abundance of hepatic amino acids in 16 h fasted male mice fed\nan HFD (n=7 mice per group, by Student’s t test). e.f, Respiratory exchange ratio (RER)\nmeasured during the dark (e) and light (f) phases by indirect calorimetry in chow-fed\nGpr146 / and Gpr146 / male mice. g,h, Magnetic resonance imaging (MRI) analysis of lean\nmass (g) and fat mass (h) of Gpr146+/+ and Gpr146-/- littermates on chow diet. * p< 0.05, ** p<\n0.01; b⁺a⁺rs in d indica⁻te⁻ mean ± s.d., bars in e-h indicate mean ± s.e.m..\n2. Regarding the energy expenditure, several questions remain. Is the increase in\ncatabolism that was observed in vivo also present in isolated adipocytes? What is the in vivo\nenergy expenditure profile in the adipose specific KOs? Does the increased energy\nexpenditure persist at thermoneutrality? How about on chow? The authors say the\nmechanism is not through canonical thermogenic cycles but Adrb3, which they show is up,\nis the major driver of both Ucp1 and non-Ucp1 dependent thermogenesis. And CL-316,243\ndoes far more than just stimulate lipolysis.\nAnswer: We thank the reviewer for these thoughtful and constructive questions. To address\nthe first point, we assessed cellular respiration using Seahorse analysis in differentiated\nhuman SGBS adipocytes following siRNA-mediated GPR146 knockdown. No significant\ndifference in oxygen consumption rate (OCR) was observed between knockdown and control\ncells (new data, Fig. R1Q2a), suggesting that the elevated energy expenditure observed in\nvivo likely involves systemic or multi-tissue interactions not recapitulated in isolated adipocytes.\nFurthermore, under HFD feeding condition, we observed a significant increase in energy\nexpenditure in both male and female mice (Fig. 3d, new data, Fig. 3g). However, under chow-\nfed conditions, we did not observe a significant difference in whole-body energy expenditure\nbetween knockout and control mice (new data, Fig. R1Q2b). Interestingly, under\nchow, knockout mice displayed a significant increase in lean mass (new data, Supplementary\nFig. 3g) and respiratory exchange ratio (RER) (new data, Supplementary Fig.3e). In adipose-\nspecific Gpr146 knockout mice subjected to HFD feeding, indirect calorimetry similarly\nrevealed no significant change in energy expenditure (new data, Supplementary Fig. 7f).\nHowever, we observed a significant increase in RER during the light (inactive) phase (new\ndata, Supplementary Fig. 7g), suggesting a shift toward greater carbohydrate utilization during\nresting conditions. This altered substrate preference may be secondary to reduced lipid flux\nfrom adipose tissue, consistent with lower circulating free fatty acid levels, and aligns with\nthe more modest reduction in body weight and adiposity observed in adipose-specific\nknockouts compared to global knockouts.\nRegarding the mechanistic interpretation, we fully agree with the reviewer that upregulation\nof Adrb3 mRNA alone is insufficient to support a definitive role for ADRB3 signalling. In male\nknockout mice, we did not observe corresponding increases in Ucp1 expression or other\ncanonical thermogenic markers, suggesting that UCP1-mediated thermogenesis is unlikely to\nunderlie the increased energy expenditure in males (Supplementary Fig. 6f, g). Interestingly,\nin female knockout mice, we observed a robust (>15-fold) increase in Ucp1 expression in\neWAT, along with upregulation of Pgc1α and Ppara (new data, Supplementary Fig. 6h, i),\nindicating potential activation of UCP1-dependent thermogenesis in females. While Adrb3 is\na known regulator of both UCP1-dependent and -independent thermogenesis, and CL-\n316,243 can elicit diverse metabolic responses, our current data are limited to mRNA-level\nchanges, which may reflect secondary or compensatory effects. We have revised the\nmanuscript accordingly to avoid overinterpretation.\nFinally, we would like to state explicitly that the precise mechanism driving the increased\nenergy expenditure in male Gpr146 knockout mice remains unknown. We agree with the\nreviewer that further investigation is required to elucidate the underlying pathways.\nFig.R1Q2, a,Seahorse Mitostress test measuring oxygen consumption rate (OCR) in SGBS\nmature adipocytes with or without GPR146 knockdown. b, Hourly energy expenditure (EE) in\nmale Gpr146 / and Gpr146 / mice fed chow (n=8 mice per group), averaged over four\nconsecutive days.\nFigure 3. GP⁺R⁺146-deficient⁻ M⁻ ice exhibit Elevated Energy expenditure. g, Indirect\ncalorimetry study in female mice fed HFD for two weeks. Hourly energy expenditure (EE) in\nfemale Gpr146 / and Gpr146 / mice fed a high-fat diet, averaged over four consecutive\ndays. Horizontal bars indicate the light (white) and dark (black) photoperiod.\nSupplementar⁺y ⁺Figure 3. GP⁻R⁻146 deficiency reprograms hepatic glucose and amino\nacid metabolism. e. Respiratory exchange ratio (RER) measured during the dark phase by\nindirect calorimetry in chow-fed Gpr146 / and Gpr146 / male mice. g, Magnetic resonance\n⁺⁺ ⁻⁻\nimaging (MRI) analysis of lean mass of Gpr146+/+ and Gpr146-/- littermates on chow diet. * p<\n0.05; bars indicate mean ± s.e.m..\nSupplementary Figure 7, Adipose GPR146 mediates protection against diet-induced\nobesity and liver steatosis, Related to Fig.4. f, Energy Expenditure measured by indirect\ncalorimetry in Adipoq-Cre and control mice (Adipoq-Cre-) fed HFD for 4 months. g, Hourly\nrespiratory exchange ratio (RER) measured in the same cohort of male Adipose specific\nknockout and the control l⁺ittermates. * p< 0.05.\nSupplementary Figure 6, Metabolic phenotyping and thermogenic pathway regulation\nin adipose tissue of GPR146-deficient and control mice, related to Fig3. h, i, qPCR\nexpression analysis of Ucp1, Pgc1a and Ppara in eWAT (h) and iWAT (i) from female\nGpr146+/+ and Gpr146-/- littermates fed HFD (n=4-8 mice per group, by Student’s t test). * p<\n0.05, ** p< 0.01; bars indicate mean± s.d.\n3. Why do the author’s think that knocking Gpr146 down in cells has an effect when they\nhaven’t added any ligand? Is it known if the ligand for GPR146 is endogenously produced?\nAnswer: This is an excellent question. There are two plausible explanations for the observed\nphenotypic effects upon GPR146 knockdown in the absence of exogenously added ligand.\nFirst, like many GPCRs, GPR146 likely exhibits constitutive (ligand-independent) basal activity,\nwhich can influence downstream signalling pathways relevant to adipocyte differentiation.\nIndeed, we found that knockdown of GPR146 in mature adipocytes reduced ERK activity,\nwhereas over-expression of GPR146 in SGBS cells significantly increased ERK activity even\nin the absence of serum supplementation (new data, Fig. 8d, supplementary Fig. 11e). This\nbasal activity has been reported for many GPCRs and may regulate intracellular signaling\neven in the absence of ligand stimulation.\nSecond, recent studies have identified Cholesin-a gut-derived hormone induced by NPC1L1-\nmediated cholesterol uptake-as an endogenous ligand for GPR1465. While we did not\nsupplement our in vitro differentiation system with Cholesin or other candidate ligands, it is\npossible that components in the culture medium (e.g., serum lipids or sterol derivatives) or\nautocrine/paracrine factors secreted by the differentiating cells themselves may serve as\nendogenous activators of GPR146.\nTogether, these two possibilities-constitutive receptor activity and potential endogenous ligand\navailability-offer a mechanistic explanation for why GPR146 knockdown leads to impaired\nadipogenesis, even without the addition of an exogenous ligand.\nFigure 8. GPR146 regulates mature adipocyte lipolysis through ERK signaling. d,\nRepresentative Western blots of phosphorylated ERK (p-ERK) and total ERK in SGBS cells\nwith or without doxycycline-induced GPR146 expression.\nSupplementary Figure 11, GPR146 regulates mature adipocyte lipolysis through ERK\nsignaling, Related to Fig. 8. e, Representative western blots of p-ERK and ERK in GPR146\nknockdown SGBS-derived mature adipocytes.\n4. All the in vivo findings involve prevention of steatosis because of constitutively expressed\ngenetic models. Inducibly depleting Gpr146 after NAFLD has developed would significantly\nstrengthen the manuscript.\nAnswer: We thank the reviewer for this important suggestion. We agree that assessing the\ntherapeutic potential of GPR146 depletion after the onset of NAFLD would be a valuable future\ndirection. While we did not perform inducible genetic deletion after NAFLD establishment, we\nadopted a complementary strategy using an AAV-delivered shRNA system to acutely deplete\nGPR146 expression in adult C57BL/6 mice together with high-cholesterol NASH diet\nchallenge (new data, Fig. 5a).\nSpecifically, mice were administered AAVs expressing shRNA targeting Gpr146, which\nresulted in approximately 40% knockdown of Gpr146 expression in both subcutaneous and\nvisceral white adipose tissues, as well as 80% in the liver (new data, Fig. 5b). Following AAV\ndelivery, mice were fed a MASH-inducing diet for 13 weeks. Notably, Gpr146 knockdown mice\nexhibited significantly lower body weight (new data, Fig. 5e), reduced adipose depot size (new\ndata, Fig. 5f, g), decreased hepatic triglyceride accumulation (new data, Fig. 5c), and reduced\ninflammatory gene expression compared to scramble shRNA controls (new data, Fig. 5d).\nThese findings indicate that acute depletion of GPR146 in adult mice is sufficient to confer\nprotection against diet-induced hepatic steatosis.\nWhile this model was used as a preventative approach, it nonetheless reinforces the\ntranslational potential of targeting GPR146 in metabolic disease and provides additional\nevidence that modulation of its activity in adulthood can yield beneficial effects on adiposity\nand hepatic lipid accumulation.\nFigure 5. AAV-mediated acute depletion of Gpr146 confirms its role in promoting\nhepatic steatosis and adiposity. a, Schematic timeline of the AAV cohort. Adult male mice\nwere injected with AAV8 expressing shRNA targeting Gpr146 (n = 9) or scramble control (n\n= 7), followed by 13 weeks of feeding with a MASH-inducing diet enriched in cholesterol and\nfructose. b, qPCR expression analysis of Gpr146 in liver, eWAT, iWAT and BAT to assess\nknockdown efficiency. c, Hepatic triglyceride content in mice with or without Gpr146\nknockdown. d, qPCR expression analysis of hepatic expression of key inflammatory genes\nin mice with or without Gpr146 knockdown. e-g, Body weight(e), iWAT(f), eWAT (g) weights\nafter 13 weeks of MASH diet feeding. * P < 0.05, **P < 0.01. bars indicate mean ± s.e.m.\nMinor\n-In the discussion, just because Ucp1 expression is unchanged does not mean the EE is\nUcp1 independent.\nAnswer: We thank the reviewer for this helpful clarification. We agree that the absence\nof Ucp1 mRNA upregulation alone is not sufficient to conclude that the increased energy\nexpenditure is UCP1-independent. Indeed, UCP1 activity may be regulated post-translational,\nor may occur in depots not sampled6,7, and we acknowledge that our current data are limited\nto gene expression analyses. We have revised the discussion accordingly to avoid\noverinterpretation and to clarify that further protein-level and functional validation will be\nrequired to determine whether UCP1 contributes to the elevated energy expenditure observed\nin GPR146-deficient male mice.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, the authors investigated the regulatory role of the G protein-coupled\nreceptor 146 (GPR146) in adipose tissue and liver lipid metabolism. In an in vivo model,\nGPR146 deficiency exhibited a protective effect against obesity induced by a high-fat diet,\nreducing liver steatosis and accompanying a decrease in circulating liver enzyme levels.\nGlobal depletion of GPR146 in mice affected energy homeostasis by increasing energy\nexpenditure during high-fat diet feeding. The study revealed that specific depletion of GPR146\nin adipocytes in mice could counteract diet-induced fatty liver by limiting the influx of free fatty\nacids. Additionally, the authors demonstrated that GPR146 deletion in vitro inhibits\nadipogenesis by blocking the activation of Gαq-PKC AKT signaling.\nThe subject matter of this study holds considerable significance in the realm of lipid\nmetabolism. Nevertheless, the data outlined in the current version do not entirely substantiate\nthe authors' conclusions. A notable deficiency lies in the absence of a mechanistic explanation\ndetailing how GPR146 influences lipid turnover and adipogenesis. Here are specific\ncomments aimed at potentially enhancing the manuscript.\n1. The first question inquires whether the protein expression level of GPR146 is elevated in\nthe adipose tissue of obese individuals compared to non-obese ones.\nAnswer: We thank the reviewer for this important question. Unfortunately, we were unable to\ndirectly assess GPR146 protein levels in adipose tissue of obese versus non-obese\nindividuals due to technical limitations. Like many GPCRs, GPR146 is expressed at low levels\nin vivo, and despite testing multiple commercially available antibodies, we found that while the\nantibody (ab151626, Abcam) effectively detects recombinantly overexpressed GPR146,\nit fails to detect endogenous protein, likely due to insufficient abundancy of endogenous\nprotein.\nHowever, at the mRNA level, we consistently observed that GPR146 expression in adipose\ntissue is inversely correlated with BMI in human cohorts (data adapted from The Adipose\nTissue Knowledge Portal8) (new data, Fig. R2Q1), and the expression is lower in obese\ncompared to non-obese individuals (new data, Supplementary Fig. 8f). We propose two\npossible explanations for this discrepancy between function and expression:\n1. Compensatory downregulation: The observed reduction in GPR146 expression in obesity\nmay represent a compensatory response to mitigate further adipose expansion or metabolic\nstress. This phenomenon has been observed in other metabolic regulators that are\ndownregulated in disease states despite their physiological function promoting those same\nprocesses.\n2. Cell-type composition shift: Obesity induces extensive adipose tissue remodeling,\ncharacterized by increased immune cell infiltration, particularly macrophages, and a\nsubstantially reduced proportion of mature adipocyte within the depot9. Because the\nexpression level of GPR146 in mature adipocytes is much higher than other cell types within\nthe adipose tissue, a relative reduction in adipocyte fraction (or increased inflammatory cell\ncontent) could result in an overall decline in measured GPR146 mRNA from bulk tissue.\nAdditionally, supporting its functional role, adipose GPR146 expression is also inversely\ncorrelated with plasma LDL-C and triglyceride levels in humans (new data, Fig. R2Q1).\nImportantly, studies from our group and others have clearly demonstrated that genetic deletion\nof Gpr146 in mice leads to significant reductions in circulating LDL-C and TG levels. These\nfindings reinforce the notion that reduced GPR146 expression is functionally protective, even\nif its mRNA levels in adipose tissue appears downregulated in obesity due to changes in tissue\ncomposition.\nFigure R2Q1. Correlation of GPR146 expression in human adipose tissue with BMI (top),\nand circulating-LDL level (bottom) based on data from the Adipose Tissue Knowledge Portal.\nSupplementary Figure 8, GPR146 expression in adipose tissue is modulated by\nnutritional and physiological context in mice and humans, Related to Fig.4. f, Forest\nplots summarizing GPR146 expression across multiple human transcriptomic cohorts (data\nadapted from the Adipose Tissue Knowledge Portal), comparing obese versus non-obese\nindividuals. Source studies included in the meta-analysis are listed alongside each plot.\n2. Does the expression level of GPR146 in wild-type mice also increase over time in various\nadipose tissues (eWAT, ingWAT, and BAT) during HFD feeding?\nAnswer: We thank the reviewer for this thoughtful question. In our analysis of wild-type mice\nsubjected to HFD feeding, we observed that Gpr146 expression was modestly decreased in\neWAT, while levels in iWAT and BAT remained largely unchanged compared to chow-fed mice\n(new data, Supplementary Fig. 8c). Consistently, publicly available datasets also reveal a\nsimilar trend, with GPR146 expression downregulated in adipose tissue of mice after HFD\nfeeding (new data, Supplementary Fig. 8d, e). This expression pattern in mouse adipose\ntissue is similar as what is observed in humans, as discussed in response to the previous\nquestion. As mentioned, we propose two possible explanations for this discrepancy between\nreduced expression and the protective phenotype associated with GPR146 deficiency. One\npossibility is that Gpr146 downregulation reflects a feedback mechanism during chronic HFD\nexposure, aimed at limiting further adipocyte expansion or lipid accumulation in metabolically\nstressed environments. Alternatively, changes in adipose tissue composition during obesity,\nincluding increased infiltration of immune and stromal cells, may reduce the relative\nabundance of Gpr146-expressing mature adipocytes, thereby contributing to the observed\ndecline in total mRNA levels from bulk tissue.\nTaken together, these observations suggest that although GPR146 expression may decline\nunder chronic obesity, its activity is likely important in regulating early adipocyte differentiation\nand lipid handling. Thus, loss of GPR146 function- either genetically or through targeted\ninhibition- can still provide metabolic protection, especially under HFD feeding.\nSupplementary Figure 8, GPR146 expression in adipose tissue is modulated by\nnutritional and physiological context in mice and humans, Related to Fig.4. c, Gpr146\nmRNA expression in eWAT, iWAT, and BAT from WT C57/BL6 mice fed a chow or high-fat\ndiet (HFD) for 3 months (n = 6–10 per group). d, e, Analysis of mouse eWAT transcriptomic\ndata from two publicly available datasets, GSE39549 (d) and GSE97271 (e), comparing\nGpr146 expression before and after HFD feeding, and its relationship with Lep (leptin)\nexpression. * p< 0.05, ** p< 0.01; bars indicate mean ± s.e.m,\n3. Global depletion of GPR146 in mice, as observed in Fig 2b by MRI measurements, led to\nan increase in the percentage of lean mass under HFD feeding. However, the data only\nindicate the percentage of lean mass. Is there an increase in the absolute amount of lean\nmass as well? Utilizing a whole-body composition analyzer might offer more precise in vivo\nmeasurements the mass of body lean, fat, and fluid.\nAnswer: We appreciate the reviewer’s suggestion regarding the assessment of absolute lean\nmass. In our study, body composition was measured using MRI, which provided both relative\nand absolute values for lean and fat mass.\nWhile we observed a significant increase in the percentage of lean mass in Gpr146 knockout\nmice under HFD feeding (Fig. 2b), the absolute lean mass did not differ significantly between\nknockout and control groups (Fig. 3a). The increased percentage is therefore a relative\nchange, driven primarily by the reduction in fat mass rather than a gain in lean tissue under\nHFD feeding.\n4. Does the protective role of GPR146 deficiency against high-fat diet (HFD)-induced obesity\nsuggest a contrasting effect when GPR146 is up-regulated? The authors should provide or\nat least discuss the results induced by GPR146 over-expressions.\nAnswer: We thank the reviewer for this thoughtful question. While it may be intuitive to expect\nthat GPR146 overexpression (OE) would elicit phenotypes opposite to those seen in GPR146\nknockout (KO) models- such as increased adiposity and worsened metabolic outcomes- our\nfindings suggest that the relationship is not strictly linear.\nIn our in vitro studies using human SGBS preadipocytes, we observed that both GPR146\nknockdown (KD) and overexpression (OE) impaired adipocyte differentiation (Fig. 6i, j, k and\nnew data, Supplementary Fig. 9h, i). This suggests that tight regulation of GPR146 expression\nis critical for proper adipogenesis, and that perturbation in either direction can be detrimental.\nThis concept is not without precedent. For example, the MAPK/ERK signalling pathway, a\nwell-studied regulator of adipogenesis, also demonstrates phase-specific and finely tuned\ncontrol during adipogenesis. ERK activation is required to initiate preadipocyte commitment,\nbut sustained ERK activation inhibits differentiation, and timely inactivation is necessary for\nlate stage of adipogenesis 10. This illustrates how precise modulation, rather than\nunidirectional activation or suppression, is essential for adipogenesis.\nWe have incorporated these new data into the revised manuscript and included a discussion\nof this observation. Taken together, our findings indicate that GPR146 expression likely needs\nto be maintained within an optimal range for normal adipocyte function. We now emphasize\nin the discussion that GPR146 modulation exhibits non-linear effects, and that both gain- and\nloss-of-function perturbations can disrupt adipogenesis, cautioning against the assumption of\nsymmetric phenotypic outcomes.\nSupplementary Figure 9, GPR146 promotes differentiation of both mouse and human\npreadipocytes, Related to Fig.6. h, Relative GPR146 mRNA expression in human SGBS\ncells following doxycycline-induced overexpression of GPR146 (n = 3 replicates per\ncondition, representative of 3 independent experiments; Student’s t test). i, Triglyceride\ncontent in SGBS-differentiated adipocytes overexpressing GPR146 or GFP (control). * p<\n0.05, ** p< 0.01; bars indicate mean ± s.e.m.\n5. Moreover, the data from GPR146 deficiency do not entirely support the authors'\nconclusions regarding the promoting effects of GPR146 on adipogenesis. To strengthen this\nassertion, the author should include data from GPR146 overexpression experiments.\nAnswer: We thank the reviewer for raising this important point. As demonstrated in our study,\nboth genetic knockout of GPR146 in mouse SVF cells and siRNA-mediated knockdown in\nhuman SGBS preadipocytes consistently resulted in impaired adipogenesis, supporting a\nphysiological role for GPR146 in promoting adipocyte differentiation (Fig.6 i-k, Supplementary\nFig.9d, e)\nAs discussed in the previous question, we also performed GPR146 overexpression\n(OE) experiments in SGBS cells. Interestingly, similar to knockdown, GPR146 OE\nalso inhibited adipogenesis, suggesting that both insufficient and excessive receptor activity\ncan disrupt the differentiation process (new data, Supplementary Fig.9h, i). These findings\nimply that GPR146 expression must be tightly regulated, and deviations in either direction can\nbe detrimental—consistent with the concept of a dose-sensitive or biphasic effect, which is not\nuncommon in GPCR signalling. That said, we place greater physiological weight on the loss-\nof-function (LOF) data in vivo, as the knockout and knockdown models show coherent\nmetabolic and cellular phenotypes under both normal and high-fat diet conditions.\nWe have now included a brief discussion of the OE findings in the revised manuscript and\nclarified that while OE results do not produce an opposite phenotype, they reinforce the\nconclusion that appropriate levels of GPR146 are critical for adipose tissue development.\nSupplementary Figure 9, GPR146 promotes differentiation of both mouse and human\npreadipocytes, Related to Fig.6. h, Relative GPR146 mRNA expression in human SGBS\ncells following doxycycline-induced overexpression of GPR146 (n = 3 replicates per\ncondition, representative of 3 independent experiments; Student’s t test). i, Triglyceride\ncontent in SGBS-differentiated adipocytes overexpressing GPR146 or GFP (control). * p<\n0.05, ** p< 0.01; bars indicate mean± s.d.,\n6. The author discussed ATP-consuming futile cycles in whole-body GPR146 deficiency. It\nraises the question whether similar findings could be observed in mice with adipocyte- or\nhepatocyte-specific GPR146 deficiency.\nAnswer: We thank the reviewer for raising this important point. We have examined the\nexpression of genes associated with ATP-consuming futile cycles in adipose tissue\nof adipocyte-specific Gpr146 knockout mice. In both eWAT and ingWAT depots, we did not\nobserve significant changes in the mRNA levels of key genes involved in lipid cycling pathways\n(new data, Fig. R2Q6a, b). Additionally, in this revision, we also performed indirect calorimetry\nin adipose-specific KO mice and found no significant difference in energy expenditure, despite\na clear reduction in fat mass (new data, Supplementary Fig. 7e, f). One possible explanation\nis that the metabolic phenotype in adipocyte-specific knockout mice is considerably smaller\nthan that observed in whole-body knockout mice (e.g., ~9% vs ~16% reduction in body weight\nafter 8 weeks of HFD feeding), which might limit our ability to detect changes at the\ntranscriptomic level.\nAs for liver-specific Gpr146 knockout mice, we did not observe any significant differences in\nbody weight or hepatic triglyceride levels under HFD feeding conditions (supplementary Fig.\n7a and Fig. 4C). Given the lack of a clear metabolic phenotype, we did not proceed with further\nanalysis of futile cycle gene expression in adipose tissue from these mice. It is worth noting,\nhowever, that our previous study11 demonstrated that GPR146 regulates plasma cholesterol\nand triglyceride levels primarily through its function in the liver, particularly via modulation of\nhepatic VLDL secretion. In contrast, the regulation of hepatic lipid accumulation in response\nto HFD feeding appears to be predominantly governed by GPR146 activity in adipose tissue,\nrather than in hepatocytes.\nWe also acknowledge the reviewer’s concern regarding our original interpretation of the futile\nlipid cycle as a mechanistic driver of increased energy expenditure. As we now clarify in the\nrevised manuscript, this conclusion was based primarily on qPCR data showing increased\nexpression of genes involved in lipid turnover and re-esterification. We agree that these\nfindings are not sufficient to establish causality, and such changes could reflect secondary\nadaptations rather than a direct mechanistic pathway. We have therefore revised the relevant\nsections to tone down the overinterpretation and relocated the discussion of lipid cycling to\nthe Discussion section, where we clearly state that the precise mechanism underlying\nincreased resting energy expenditure in male Gpr146 knockout mice remains unresolved.\nIn our revised study, we also performed indirect calorimetry in female GPR146-deficient mice,\nand found that KO females exhibited significantly increased energy expenditure compared to\ncontrols during both light and dark cycles (new data, Fig. 3g). Interestingly, in contrast to male\nKO mice where Ucp1 expression remained unchanged in eWAT and ingWAT (Supplementary\nFig. 6f, g), female KO mice displayed a more than 15-fold increase in Ucp1 expression\nspecifically in eWAT, along with marked upregulation of Pgc1α and Ppara (new data,\nSupplementary Fig. 6h, i). These data suggest that in females, activation of UCP1-mediated\nthermogenesis may contribute to the observed increase in energy expenditure, potentially\nthrough enhanced mitochondrial biogenesis and oxidative metabolism. This emerging sex\ndifference in thermogenic activation may reflect differential hormonal regulation or adipose\ntissue plasticity between males and females, and further investigation to elucidate sex-specific\nmechanisms underlying GPR146-dependent metabolic control is required.\nTo further explore the role of acute GPR146 depletion, we performed an AAV-shRNA-\nmediated knockdown of Gpr146 in wild-type mice. This approach led to efficient knockdown\nin the liver (~80% mRNA reduction) and moderate knockdown (~40%) in white adipose tissues\n(new data, Fig. 5b). Notably, mice with Gpr146 depletion via AAV-shRNA\nexhibited significantly reduced body weight, decreased eWAT and ingWAT depot sizes, and\nlower hepatic triglyceride content under HFD feeding (new data, Fig. 5e, f, g, c). These results\nsupport the notion that partial and acute reduction of GPR146 in both liver and adipose\ntissue is sufficient to confer metabolic benefits, and align well with the phenotypes observed\nin our genetic knockout models.\nFig.R2Q6. a,b. Relative mRNA expression of genes involved in lipid cycling in iWAT (a) and\neWAT (b) from adipo-specific knockout mice and their control littermates fed HFD for 4\nmonths. * p< 0.05, ** p< 0.01; bars indicate mean± s.d.,\nSupplementary Figure 7, Adipose GPR146 mediates protection against diet-induced\nobesity and liver steatosis, Related to Fig.4. e, MRI analysis of fat mass and lean mass\nfrom female adipose-specific knockout mice (Adipoq-Cre+) and control littermates (Adipoq-\nCre-) fed HFD for 4 months. f, Energy Expenditure measured by indirect calorimetry in\nAdipoq-Cre and control mice (Adipoq-Cre-) fed HFD for 4 months. * p< 0.05, ** p< 0.01;\nbars in all panels indicate mean± s.d..\nFigure 3. G⁺PR146-deficient Mice exhibit Elevated Energy expenditure. g, Indirect\ncalorimetry study in female mice fed HFD for two weeks. Hourly energy expenditure (EE) in\nfemale Gpr146 / and Gpr146 / mice fed a high-fat diet, averaged over four consecutive\ndays. Horizontal bars indicate the light (white) and dark (black) photoperiod (n=8 mice per\ngroup). ⁺ ⁺ ⁻ ⁻\nSupplementary Figure 6, Metabolic phenotyping and thermogenic pathway regulation\nin adipose tissue of GPR146-deficient and control mice, Related to Fig3. h,i, qPCR\nexpression analysis of Ucp1, Pgc1a and Ppara in eWAT (h) and iWAT (i) from female\nGpr146+/+ and Gpr146-/- littermates fed HFD (n=4-8 mice per group, by Student’s t test). * p<\n0.05, ** p< 0.01; bars in h and i indicate mean± s.d.; bars in f and g indicate mean ± s.e.m..\nFigure 5. AAV-mediated acute depletion of Gpr146 confirms its role in promoting\nhepatic steatosis and adiposity. b, qPCR expression analysis of Gpr146 in liver, eWAT,\niWAT and BAT to assess knockdown efficiency. c, Hepatic triglyceride content in mice with\nor without Gpr146 knockdown. e-g, Body weight (e), iWAT(f), eWAT (g) weights after 13\nweeks of MASH diet feeding. * P < 0.05, **P < 0.01. bars indicate mean ± s.e.m.\n7. Since PKC is the endpoint of the proposed GPR146 signaling pathway, in the GPR146\ndepletion background, inhibition of PKC function by pharmacological reagent alone cannot\nsupport the authors' hypothesis that Gαq-PKC-pAkt is downstream of GPR146 signaling.\nAnswer: We thank the reviewer for their thoughtful question. However, we would like to clarify\nthat in our proposed signalling pathway, AKT (measured as p-AKT), not PKC, is the\ndownstream endpoint. Our hypothesis states that GPR146 activates Gαq, which in turn\nactivates PKC, leading to the phosphorylation of AKT (p-AKT), a key effector in subcutaneous\nadipogenesis. Thus, we assess pathway activity by measuring p-AKT levels, not PKC directly.\nIn our experiments, GPR146 knockdown (KD) pre-adipocytes exhibited reduced p-AKT levels,\nindicating that the pathway is compromised when GPR146 is depleted. When we treated these\nKD cells with a PKC inhibitor, we observed no further reduction in p-AKT or adipogenesis,\nunlike in control cells where the inhibitor significantly decreased both. This lack of additive\neffect in KD cells supports our model: GPR146 depletion upstream already disrupts the Gαq-\nPKC-pAkt cascade, so inhibiting PKC downstream has no additional impact. In contrast, PKC\ninhibition in control cells confirms its role in the intact pathway (Fig. 7f, g, h).\nTherefore, our use of p-AKT as a readout, combined with the PKC inhibitor experiments,\ndirectly supports the hypothesis that Gαq-PKC-pAkt operates downstream of GPR146.\nWe hope this clarifies that AKT, not PKC, is the endpoint we are evaluating.\n8. To address the roles of GPR146 in lipid turnover, the authors should provide more\nfunctional experiments, instead of just differential expressions analyses.\nAnswer: We appreciate the reviewer’s suggestion to include additional functional evidence\nbeyond transcriptomic analyses to support our claim that GPR146 regulates lipid turnover.\nWe fully agree that our initial interpretation may have overstated the mechanistic link between\nGPR146 deficiency and increased lipid turnover, particularly given that the original conclusion\nwas based primarily on differential gene expression. Honestly, while we present robust data\ndemonstrating that adipose-specific depletion of GPR146 attenuates both adipogenesis and\nlipolysis-thereby reducing FFA efflux and contributing to protection against hepatic steatosis-\nthe precise molecular mechanism by which GPR146 deficiency enhances energy expenditure\nremains unclear in male mice. Although we initially speculated the involvement of ATP-\nconsuming futile cycles, our evidence is limited to mRNA expression data. In the revised\nmanuscript, we have moved this hypothesis into the Discussion section and clearly\nacknowledged the current limitations in direct mechanistic evidence.\nIn our revised study, we also performed indirect calorimetry in female GPR146-deficient mice,\nand found that KO females exhibited significantly increased energy expenditure compared to\ncontrols during both light and dark cycles (new data, Fig. 3g). Interestingly, in contrast to male\nKO mice where Ucp1 expression remained unchanged in eWAT and ingWAT (Supplementary\nFig. 6f, g), female KO mice displayed a more than 15-fold increase in Ucp1 expression\nspecifically in eWAT, along with marked upregulation of Pgc1α and Ppara (new data,\nSupplementary Fig. 6h, i). These data suggest that in females, activation of UCP1-mediated\nthermogenesis likely contribute to the observed increase in energy expenditure, potentially\nthrough enhanced mitochondrial biogenesis and oxidative metabolism. This emerging sex\ndifference in thermogenic activation may reflect differential hormonal regulation or adipose\ntissue plasticity between males and females1,2, and further investigation to elucidate sex-\nspecific mechanisms underlying GPR146-dependent metabolic control is required.\nImportantly, we would like to highlight the systemic metabolic reprogramming in GPR146-\ndeficient mice. The knockout mice exhibit improved glucose handling, accompanied\nby increased hepatic glycogen content and elevated levels of hexose and pentose phosphate\nintermediates, suggesting a possible enhancement in hepatic insulin sensitivity (Fig. 2k, l and\nSupplementary data Fig. 3a, b, c). The accumulation of pentose phosphate intermediates\nfurther suggests increased NADPH production3, which could contribute to redox\nhomeostasis and protection from oxidative stress, a key driver of hepatic inflammation in\nNAFLD4.\nAdditionally, untargeted metabolomics also revealed significantly elevated hepatic levels of\nseven standard amino acids in Gpr146-/- mice, namely histidine, cysteine, lysine, valine,\nleucine, isoleucine, and phenylalanine (new data, Supplementary Fig. 3d). Consistent with\nthese hepatic metabolic changes, chow-fed Gpr146-/- mice exhibited a significantly higher\nrespiratory exchange ratio (RER) during the dark cycle compared to controls (new data,\nSupplementary Fig. 3e, f), indicating a greater reliance on glucose as an energy source.\nFurthermore, chow-fed Gpr146-/- mice displayed significantly increased lean mass without\nchanges in fat mass (new data, Supplementary Fig. 3g, h), potentially reflecting altered\nsystemic nutrient partitioning and increased availability of amino acids at chow-fed baseline\ncondition. Together, these findings support a model in which GPR146 deficiency induces\ncoordinated metabolic shifts that reduce the need for lipid storage and limit ectopic lipid\naccumulation in the liver.\nTo directly probe the role of GPR146 in mature adipocyte where it is most highly expressed,\nwe performed complementary in vitro and in vivo lipolysis assays.\nIn mature SGBS-derived adipocytes, we found that doxycycline-induced GPR146\noverexpression significantly increased lipolysis, as measured by glycerol release (new data,\nFig. 8c). Furthermore, we demonstrated that this increase in lipolysis was dependent on ERK\nsignalling as treatment with the MEK1 inhibitor PD0325901 attenuated the lipolysis response\n(new data, Fig. 8c). In contrast, siRNA-mediated knockdown of GPR146 in SGBS adipocytes\nsignificantly reduces lipolytic activity (new data, Fig. 8b).\nTo assess physiological relevance, we utilized an AAV-shRNA-based knockdown approach to\ntransiently deplete GPR146 in vivo in mice fed a NASH diet. GPR146 knockdown led to\nsignificant reductions in body weight, adipose depot mass, and hepatic triglyceride levels,\nclosely mirroring the phenotypes observed in our constitutive knockout models (new data, Fig.\n5c, e, f, g). Importantly, upon CL-316,243 stimulation, glycerol release was reduced in\nGPR146 knockdown mice compared to controls, suggesting diminished lipolytic capacity,\nwhich likely contributes to the observed reduction in hepatic TG accumulation (new data, Fig.\n8e).\nWhile these new findings do not establish a direct role for GPR146 in driving lipid turnover\nthrough a futile cycle, they support a functional link between GPR146 activity in adipose tissue\nand hepatic lipid homeostasis, particularly by modulating lipolysis and FFA availability to the\nliver. We have incorporated these new data into the revised manuscript to address the\nreviewer’s comment and strengthen our conclusions.\nFigure 3. GPR146-deficient Mice Have Elevated Energy expenditure during Light\nPhotoperiod. g, Indirect calorimetry study in female mice fed HFD for two weeks. Hourly\nenergy expenditure (EE) in female Gpr146 / and Gpr146 / mice fed a high-fat diet, averaged\nover four consecutive days. Horizontal bars indicate the light (white) and dark (black)\nphotoperiod (n=8 mice per group). * p< 0.0⁺5⁺, ** p< 0.01. ⁻ ⁻\nSupplementary Figure 6, Metabolic phenotyping and thermogenic pathway regulation\nin adipose tissue of GPR146-deficient and control mice, related to Fig3. h,i, qPCR\nexpression analysis of Ucp1, Pgc1a and Ppara in eWAT (h) and iWAT (i) from female\nGpr146+/+ and Gpr146-/- littermates fed HFD (n=4-8 mice per group, by Student’s t test). * p<\n0.05, ** p< 0.01; bars in h and i indicate mean± s.d.; bars in f and g indicate mean ± s.e.m..\nSupplementary Figure 3. GPR146 deficiency reprograms hepatic glucose and amino\nacid metabolism. d, Relative abundance of hepatic amino acids in 16 h fasted male mice\nfed an HFD (n=7 mice per group, by Student’s t test). e.f, Respiratory exchange ratio (RER)\nmeasured during the dark (e) and light (f) phases by indirect calorimetry in chow-fed\nGpr146 / and Gpr146 / male mice. g,h, Magnetic resonance imaging (MRI) analysis of\nlean mass (g) and fat mass (h) of Gpr146+/+ and Gpr146-/- littermates on chow diet. * p< 0.05,\n** p< 0⁺.⁺01; bars in a-d⁻ ⁻indicate mean ± s.d., bars in e-h indicate mean ± s.e.m..\nFigure 8. GPR146 regulates mature adipocyte lipolysis through ERK signaling. b.\nGlycerol release from SGBS mature adipocytes transfected with siGPR146 or control siRNA,\nwith or without forskolin stimulation; values normalized to intracellular triglyceride (n=3\nreplicates per experiment, representative of 3 independent experiments, by Student’s t test).\nc, Glycerol release from SGBS adipocytes with doxycycline-inducible GPR146 expression.\nMeasurements were performed with or without the MEK inhibitor PD98059 and normalized\nto intracellular TG (n=3 replicates per experiment, representative of 3 independent\nexperiments, by Student’s t test). e, In vivo lipolysis assay following CL-316,243 stimulation\nin mice injected with AAV-shGpr146 or scramble control shRNA and maintained on a MASH-\ninducing diet for 6 weeks. Plasma glycerol was measured at the indicated time points upon\nCL316,243 injection. * p< 0.05, ** p< 0.01; bars indicate mean± s.e.m..\nFigure 5. AAV-mediated acute depletion of Gpr146 confirms its role in promoting\nhepatic steatosis and adiposity. c, Hepatic triglyceride content in mice with or without\nGpr146 knockdown. e-g, Body weight(e), iWAT(f), eWAT (g) weights after 13 weeks of\nMASH diet feeding. * P < 0.05, **P < 0.01. bars indicate mean ± s.e.m.\nMinor points:\n1. It would be helpful to provide a brief description of G protein-coupled receptors in\nmediating functional activities of adipose tissues.\nAnswer: Among the molecular pathways orchestrating adipose tissue function, G protein–\ncoupled receptors (GPCRs) play a central role in regulating lipid metabolism, adipogenesis,\nand systemic energy balance12. Several GPCRs expressed in adipose tissue, such as the\ncannabinoid receptor CB1, have been shown to promote hepatic steatosis by increasing\nadipocyte lipogenesis and FFAs released into circulation. Conversely, adipose-specific\ndeletion of CB1 protects against hepatic lipid accumulation. These findings highlight the\nessential role of GPCR-mediated adipose signaling in coordinating systemic lipid flux and\nposition adipose-resident GPCRs as important contributors to MASLD pathophysiology.\n.\nWe’ve included this paragraph in the revised manuscript.\n2. The image should include a scale bar for reference.\nAnswer: We’ve included scale bar as suggested.\n3. Please include the sequences for the siRNA used.\nAnswer: siRNA sequences were provided as suggested.\nReviewer #3 (Remarks to the Author):\nThe study by Shi et al. provides a large set of data investigating the effects of GPR146 on liver\nsteatosis and particularly the role of adipocyte GPR146. The authors first describe findings\nextracted from public GWAS databases indicating association of GPR146 variants with\nincreased plasma liver enzymes and CRP. In the following experiments they show that\nGPR146 knockout reduces liver steatosis and plasma ALT in mice. Liver metabolomics was\nused to characterize in more detail effects on lipids. GPR146 knockout also decreased weight\ngain and fat mass in response to HFD that appears to be related to elevated energy\nexpenditure during resting periods. The authors used tissue specific knockout models to\ndemonstrate that Adipoq-Cre but not Alb-Cre induced knockout alleviated the liver steatosis\nsuggesting that adipocyte GPR146 mediates the steatotic effect. Adipocyte KO of GPR146\nalso reduced weight gain, fat mass and plasma FFA. In cell models GRP146 was found to\npromote adipogenesis potentially through Gαq-PCK-AKT pathway. The study is interesting\nand thorough. However, there are several points of concern:\n1. The effect of Adipoq-Cre induced knockout on Grp146 expression in WAT requires further\ninformation. At least after 4 months of HFD the effect of the KO on Gpr146 expression appears\neither limited (ingWAT) or not existing (eWAT) (Extended Figure 7e). Please provide\nnecessary evidence of the effect of Grp146 KO on GPR146 expression in different time points\nused in the study. The current data raises questions about the mechanisms of the observed\neffects in Adipoq-KO mice.\nAnswer: We thank the reviewer for this important observation. We agree that the limited\nGpr146 depletion in eWAT after 4 months of HFD feeding in the Adipoq-Cre–driven adipose-\nspecific knockout model raises valid questions about the interpretation of the observed\nphenotypes.\nWe acquired the Adipoq-Cre line from The Jackson Laboratory, and proper genotyping was\nconfirmed prior to initiating experiments. In young adult mice (8 weeks old) maintained on\nchow diet, we validated that Gpr146 expression was significantly reduced across multiple\nadipose depots, including iWAT, eWAT, and BAT, but not in the liver and muscle, in Adipoq-\nCre+;Gpr146fl/fl mice compared to Cre-negative littermate controls (new data, Supplementary\nFig. 7d). This confirms that Cre-mediated recombination is effective.\nHowever, as the reviewer correctly noted, after prolonged HFD feeding (approximately 4\nmonths), Gpr146 expression in eWAT was no longer significantly different between knockout\nand control mice (Supplementary Fig. 7c), whereas iWAT still showed consistent and\nsignificant depletion. The reason for this apparent loss of knockdown efficiency over time in\neWAT is not fully understood. One possible explanation is the regional differences in Cre\nrecombination efficiency under prolonged metabolic stress. Additionally, prolonged HFD\nfeeding is known to cause substantial changes in adipose tissue composition where there is\nmarked infiltration of immune cells and a corresponding reduction in the proportion of mature\nadipocytes9—the cell type in which Adipoq-Cre is active. This deficit in mature adipocytes\ncould dilute the overall depletion effect observed at the mRNA level when analysing bulk tissue.\nWe acknowledge that this temporal limitation complicates the interpretation of long-term\nadipose-specific KO phenotypes. As such, we have revised the manuscript to discuss this\ncaveat and have more cautiously interpreted the Adipoq-Cre results.\nSupplementary Figure 7, Adipose GPR146 mediates protection against diet-induced\nobesity and liver steatosis, Related to Fig.4. d, Relative expression of Gpr146 mRNA in\niWAT, eWAT, BAT, liver and muscle from male mice at 8-week-old (n=8 mice per group, by\nStudent’s t test). * p< 0.05, ** p< 0.01; bars indicate mean± s.d..\n2. The authors state in several instances that GRP146 KO ameliorates hepatic inflammation.\nHowever, I don’t think any evidence is presented to support this notion. ALT cannot be\nconsidered and an inflammation marker. However, indeed it would be necessary to study the\nrole of GPR146 in relation to liver inflammation and fibrosis. Simple HFD model may not be\nthe best for this purpose, but it may require additional factors such as fructose feeding. The\nauthors also reported elevated CRP associated with GPR146 variants in humans. What\nabout in mice? Does the Gpr146 KO affect CRP?\nAnswer: We thank the reviewer for highlighting this important point. We agree that ALT is a\nmarker of hepatocellular injury, but not a specific indicator of hepatic inflammation. In our study,\nwe performed transcriptomic analysis of liver tissue from both male and female Gpr146\nknockout and control mice under HFD conditions. Gene set enrichment analysis\n(GSEA) revealed significant downregulation of multiple inflammation-related pathways,\nincluding inflammatory response, TNFα signaling via NF-κB, and interferon-γ response, in\nGpr146-deficient mice compared to controls. These findings suggest a reduction in hepatic\ninflammatory tone in the absence of GPR146 (Fig. 1l ,m).\nTo further support this observation, we evaluated inflammatory gene expression in an\nindependent model using AAV-shRNA–mediated knockdown of Gpr146 in adult mice fed\na high-cholesterol, fructose-containing NASH diet, which better models liver inflammation 13.\nIn this model, we observed significant reductions in hepatic mRNA levels of key pro-\ninflammatory cytokines, including Tnfa , Il1β, and Il6, in Gpr146 knockdown mice relative to\ncontrols (new data, Fig. 5d). While these findings support a consistent pattern of reduced\ninflammatory gene expression, we recognize the reviewer’s concern regarding\noverinterpretation. Accordingly, we have carefully revised the manuscript to more precisely\nstate that GPR146 depletion protects against hepatic triglyceride accumulation, and we now\nexplicitly clarify that our data demonstrate a downregulation of inflammatory gene expression,\nrather than direct evidence of reduced hepatic inflammation.\nRegarding CRP, while our human eQTL analysis suggests that GPR146 variants are\nassociated with altered CRP levels 14, we did not observe a consistent change in\nhepatic Crp mRNA level or plasma CRP concentration in Gpr146 knockout mice under HFD\nconditions. This may reflect species-specific differences in CRP regulation, as CRP is a much\nstronger acute-phase reactant in humans than in mice (new data, Fig. R3Q2 a, b).\nFigure 5. AAV-mediated acute depletion of Gpr146 confirms its role in promoting\nhepatic steatosis and adiposity. d, qPCR expression analysis of hepatic expression of key\ninflammatory genes in mice with or without Gpr146 knockdown. * P < 0.05, **P < 0.01. bars\nindicate mean ± s.e.m.\nFig.R3Q2. a, Relative expression of Crp in liver of Gpr146+/+ and Gpr146-/- mice littermates\nfed HFD for 3 months (n=4 per group, by Student’s t test). b, Plasma concentration of CRP\nestimated by CPR ELISA. * P < 0.05, **P < 0.01. bars indicate mean ± s.e.m.\n3.The effect on glucose metabolism should be better explained. Do the increased liver hexose\nmetabolites indicate upregulation of hepatic glucose uptake or what is the authors’\ninterpretation? It is not clear what the authors mean by statement that “GRP146 deficiency\npromotes glucose mobilization”, page 5 line 9-10. Indeed, glycogen storage was increased.\nAnswer: We thank the reviewer for this insightful comment and the opportunity to clarify our\ninterpretation regarding glucose metabolism in GPR146-deficient mice. The original statement\nthat “GPR146 deficiency promotes glucose mobilization” was imprecise, and we agree it could\nbe misinterpreted as an increase in glucose output or utilization. We have revised this in the\nmanuscript to improve clarity and accuracy.\nIn our study, Gpr146 knockout mice exhibited significantly improved glucose and insulin\ntolerance tests, consistent with enhanced systemic insulin sensitivity. In the liver, we observed\na notable increase in glycogen content as well as elevated levels of hexose and pentose\nphosphate intermediates. While we cannot conclusively determine whether the glycogen\naccumulation results from increased glucose uptake or reduced glucose oxidation, these\nfindings suggest that carbohydrate flux is redirected toward storage in the liver, likely attributed\nto enhanced hepatic insulin sensitivity in the absence of GPR146.\nTo further explore this, we performed indirect calorimetry in chow-fed mice and found that\nGPR146-deficient mice exhibited significantly enhanced respiratory exchange ratio (RER)\n(new data, Supplementary Fig. 3e), suggesting a greater reliance on carbohydrates as an\nenergy source. When considered alongside the increased hepatic glycogen content\n(Supplementary Fig. 3b, c) and significantly increased lean mass (new data, Supplementary\nFig. 3g), these findings suggest that, in GPR146-KO mice under chow, nutrient partitioning is\nreprogrammed: glucose is preferentially used for energy and lean tissue maintenance rather\nthan contributing to lipogenesis in liver and adipose tissue. We propose that this metabolic\nshift results from a combination of enhanced hepatic insulin sensitivity and a defect in\nadipogenesis, leading to reduced lipid storage capacity and altered systemic energy allocation.\nThis metabolic profile is consistent with improved hepatic insulin action, which drives glucose\nstorage as glycogen and promotes diversion of glucose into the pentose phosphate pathway.\nImportantly, the accumulation of pentose phosphate pathway intermediates suggests\nincreased NADPH production (Supplementary Fig. 3a), which may serve a cytoprotective role\nby maintaining redox homeostasis and mitigating oxidative stress. Given the well-established\nlink between oxidative stress and hepatic inflammation in NAFLD progression, we think that\nenhanced PPP flux may contribute to the anti-inflammatory and hepatoprotective\neffects observed in GPR146-deficient mice.\nThis metabolic shift—characterized by reduced lipid accumulation, enhanced glucose storage,\nand increased redox buffering capacity—combined with diminished FFA influx into the liver,\noffers a coherent explanation for the observed reduction in hepatic triglycerides, improved\ninsulin sensitivity, and attenuated inflammatory gene expression. We have revised the\nmanuscript accordingly to reflect this mechanistic insight and thank the reviewer for prompting\nthis clarification.\nSupplementary Figure 3. GPR146 deficiency reprograms hepatic glucose and amino\nacid metabolism. e., Respiratory exchange ratio (RER) measured during the dark phase by\nindirect calorimetry in chow-fed Gpr146 / and Gpr146 / male mice. g, Magnetic resonance\nimaging (MRI) analysis of lean mass of Gpr146+/+ and Gpr146-/- littermates on chow diet. * p<\n0.05, ** p< 0.01; bars indicates mean ⁺± ⁺s.e.m.. ⁻⁻\n4. The authors claim that GRP146 KO ameliorates insulin resistance. However, this is not\nobvious. The KO improves glucose tolerance (fig. 2g) which could suggest better insulin\ntolerance. However, in ITT the effects can be seen only in late time points when the animals\nare recovering from the insulin effect. A true difference in insulin sensitivity should be seen in\nearly time points when glucose is decreasing. This is especially obvious in female mice\nwhere the effect in ITT is observed only after 90 min (Ex. Fig. 3d) but also to some extent in\nmale mice (Fig 2h). The results rather appear to suggest defects in counter-regulatory\nmechanisms correcting insulin-induced hypoglycemia. Also, there was no difference in\nplasma fasting insulin (Ex. Fig. 3e) again suggesting no clear effect on insulin sensitivity.\nHOMA-IR index, preferably calculated with the mouse modified formula (doi:\n10.1177/0023677212473714) could be provided also.\nAnswer: We appreciate the reviewer’s critical evaluation of our claim regarding insulin\nsensitivity. The improved glucose tolerance observed in Gpr146 knockout (KO) mice (Fig. 2k)\ninitially suggested enhanced insulin responsiveness. However, we agree that the insulin\ntolerance test (ITT) results do not clearly support this interpretation. As the reviewer rightly\npointed out, the differences in blood glucose during ITT are primarily observed during the late\nrecovery phase (after 60–90 minutes), particularly in female mice (Supplementary Fig. 5b) and\nto some extent in males (Fig. 2l), rather than during the early glucose-lowering phase where\ntrue insulin sensitivity is best assessed. Consistently, glucose tolerance tests (GTT) showed\na similar temporal pattern: Gpr146 knockout mice exhibited lower blood glucose levels mainly\nduring the later stages of the test (Fig. 2k and Supplementary Fig. 5a), without significant\ndifferences in the early glucose clearance phase. Taken together, these findings point toward\nimproved hepatic glucose handling and metabolic flexibility in Gpr146-deficient mice,\nrather than a direct enhancement of peripheral insulin sensitivity15.\nFurthermore, we observed no significant differences in fasting insulin levels between Gpr146\nKO and control mice (Supplementary Fig. 5d), and this is consistent with the possibility that\nsystemic insulin sensitivity is not markedly improved. Following the reviewer’s suggestion,\nwe have now calculated the HOMA-IR index using fasting glucose and insulin levels, applying the\nmouse-specific formula 16. These results are now included in the revised manuscript (new data,\nSupplementary Fig. 5e) to provide an additional and more robust assessment of insulin\nresistance.\nIn light of these data, we have revised our manuscript to avoid overclaiming improved insulin\nsensitivity. Instead, we now describe the metabolic phenotype of Gpr146 KO mice as reflecting\na complex reprogramming of hepatic and systemic metabolism. Notably, liver metabolomics\nrevealed increased glycogen storage, reduced lipid species (including FFAs and long-chain\nacyl-CoAs), and elevated pentose phosphate intermediates, suggesting a shift toward glucose\nstorage and redox balancing (Fig. 1h, i, and supplementary Fig. 3a-c).\nIn support of this view, transient Gpr146 knockdown mice showed reduced glycerol release in\nresponse to CL-316,243 stimulation, indicating diminished lipolytic activity (new data, Fig. 8e).\nWe hypothesize that this reduction in circulating glycerol may impair hepatic gluconeogenesis,\ncontributing to the blunted counter-regulatory response during the ITT recovery phase.\nTaken together, these findings suggest that GPR146 deficiency induces a metabolic state\ncharacterized by altered glucose handling, reduced substrate availability for gluconeogenesis,\nand a preference for non-lipid oxidative pathways, rather than classical improvements in\ninsulin sensitivity. We have revised the manuscript accordingly to reflect this interpretation and\nthank the reviewer for prompting this clarification.\nSupplementary Figure 5. GPR146 deficiency alters systemic glucose metabolism,\nRelated to Fig.2. e, HOMA-IR calculated from fasting glucose and insulin levels in mice fed\nHFD for 1 month (n = 8–11 per group; Student’s t-test). * p< 0.05, ** p< 0.01; bars in a, b\nand e indicate mean± s.e.m.; bars in d indicate mean ± s.d..\nFigure 8. GPR146 regulates mature adipocyte lipolysis through ERK signaling. e, In\nvivo lipolysis assay in mice with acute depletion of Gpr146 using AAV-shRNA or scramble\ncontrol, maintained on a MASH-inducing diet for 6 weeks. Mice were injected with CL-\n316,243 to stimulate lipolysis, and plasma glycerol levels were measured at the indicated\ntime points. (n=7-9 mice per group, by Student’s t test). * p< 0.05, ** p< 0.01; bars indicate\nmean± s.e.m..\n5. The authors provide transcriptomics data suggesting decreased macrophage infiltration in\nthe adipose deposits of the GRP146 KO mice. Is this supported by any other data such as\nhistology?\nAnswer: As shown in the H&E staining of both iWAT and eWAT (new data, Fig. 2i, j)\nshowed minimal immune cell infiltration in GPR146 KO mice, consistent with a lower\ninflammatory burden. In contrast, crown-like structures—indicative of macrophage\naccumulation around dead adipocytes—were occasionally observed in control mice. While\nwe did not perform immunohistochemical staining for specific macrophage markers, the\nconcordance between histological observations and transcriptomic signatures strengthens\nthe conclusion that GPR146 deficiency is associated with reduced adipose tissue\ninflammation.\nFigure 2. GPR146 Promotes HFD-induced Obesity in Mice. i,j, Representative images of\n(H&E)-stained eWAT with Crown-like structure (CLS) indicated by red arrows (i).\nQuantification of CLSs per 100 adipocytes (j). * p< 0.05, ** p< 0.01; bars in indicate mean±\ns.e.m.\n6. The study includes several types of omics data; however, the full data has not been made\navailable and no repository for data storage has been indicated?\nAnswer: We have deposited the microarray data in GEO and are currently awaiting the\nassignment of an accession number, which will be provided once available.\nReferences:\nFunction: Sex Hormones, Glucocorticoids, and Their Crosstalk. Front Endocrinol\n(Lausanne) 12, 652444 (2021). https://doi.org/10.3389/fendo.2021.652444\ncardiometabolic health: the role of adipose tissue, muscle and liver. Nat Rev\nEndocrinol 17, 47-66 (2021). https://doi.org/10.1038/s41574-020-00431-8\npentose phosphate pathway. Biol Rev Camb Philos Soc 90, 927-963 (2015).\nhttps://doi.org/10.1111/brv.12140\npathogenesis of nonalcoholic fatty liver disease. Free Radic Biol Med 152, 116-141\n(2020). https://doi.org/10.1016/j.freeradbiomed.2020.02.025\n1685-1700 e1618 (2024). https://doi.org/10.1016/j.cell.2024.02.024\nAdipose Tissue by Reversible Succinylation. Mol Cell 74, 844-857 e847 (2019).\nhttps://doi.org/10.1016/j.molcel.2019.03.021\nand sulfenylation of UCP1. Nature 532, 112-116 (2016).\nhttps://doi.org/10.1038/nature17399\nexperimental data from human adipose tissue. Cell Metab 37, 566-569 (2025).\nhttps://doi.org/10.1016/j.cmet.2025.01.012\nweight loss. Nature 644, 769-779 (2025). https://doi.org/10.1038/s41586-025-09233-\n2\ndifferentiation and obesity. Biochimie 87, 51-56 (2005).\nhttps://doi.org/10.1016/j.biochi.2004.10.018\nAtherosclerosis. Cell 179, 1276-1288 e1214 (2019).\nhttps://doi.org/10.1016/j.cell.2019.10.034\nreceptor signaling in obesity and type 2 diabetes. FEBS J 288, 2622-2644 (2021).\nhttps://doi.org/10.1111/febs.15800\nNonalcoholic Steatohepatitis. Cell Metab 24, 848-862 (2016).\nhttps://doi.org/10.1016/j.cmet.2016.09.016\nCardiometabolic Risk Profile. Arterioscler Thromb Vasc Biol 42, 1262-1271 (2022).\nhttps://doi.org/10.1161/ATVBAHA.122.317514\nphenotyping in diabetes research. Diabetologia 61, 526-538 (2018).\nhttps://doi.org/10.1007/s00125-017-4495-9\n16 van Dijk, T. H. et al. A novel approach to monitor glucose metabolism using stable\nisotopically labelled glucose in longitudinal studies in mice. Lab Anim 47, 79-88\n(2013). https://doi.org/10.1177/0023677212473714"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I want to commend the authors are giving a strong rigorous effort to addressing my concerns and questions. Overall, I am quite satisfied. A few points remain for clarification:\n-What is happening to the RER at the end of the Supp Figure 7 panel g?\n-Quantification of the western blots would be beneficial\n-Figure 3g-i has the EE for the females corrected by bodyweight. This should be changed to the data without bodyweight correction for accurate representation like the male data in the first part of Figure 3\n(https://pubmed.ncbi.nlm.nih.gov/40993210/)\n-Following up on the EE, I suppose the adipose KO mice in indirect calorimetry, paired with the cell data on isolated adipocytes, would suggest the EE is not arising from the fat tissue?", "reviewer_2": "In this revised version of the manuscript, the authors have made considerable efforts to strengthen the study by incorporating a substantial amount of new experimental data. The work now presents a more comprehensive and coherent narrative, encompassing large-scale data analyses, mouse experiments, and mechanistic investigations. Although some results do not fully align with the initial expectations, the authors have provided reasonable explanations to address these discrepancies. I have no further questions or concerns regarding this version of the manuscript.", "reviewer_3": "The authors have well addressed my concerns. No further comments."}, "author_response": "Point-by-Point Response\nWe sincerely thank the reviewers for their thoughtful evaluation of our revised\nmanuscript and for the constructive comments provided. In response, we have\ncarefully revised the manuscript and addressed all remaining concerns with additional\ndata, new analyses, and further clarifications where appropriate.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI want to commend the authors are giving a strong rigorous effort to addressing my\nconcerns and questions. Overall, I am quite satisfied. A few points remain for\nclarification:\n-What is happening to the RER at the end of the Supp Figure 7 panel g?\nAnswer: We thank the reviewer for pointing this out. We also noticed the unusual\nincrease in RER during the final 12-hour interval of the measurement (84-96 h in\nSupplementary Fig. 7g). We thoroughly investigated this by checking with the\nmetabolic core facility, and they confirmed that no technical issues occurred during the\nrecording period. For additional transparency, we now provide the raw VO₂ and VCO₂\ntraces (Figure a and b), which show consistent patterns, and no abrupt spikes were\ndetected in locomotor or ambulatory activity (Figure c and d), suggesting that the RER\nelevation was not due to increased exploratory behaviour or physical activity.\nImportantly, excluding the final 12-h interval does not affect our main\nconclusion: Adipoq-Cre⁺ mice exhibit a significantly higher RER than controls during\nthe light (resting) phase, which remains robust even without this final segment. We\nhave clarified this point in the revised figure legend and ensured that this anomalous\ntime point does not bias the interpretation of our data.\nFigure (a-d): Oxygen consumption (a), carbon dioxide production (b), locomotor activity (c)\nand Ambulatory activity (d) measured by indirect calorimetry in Adipoq-Cre⁺ and control mice\n(Adipoq-Cre-) fed HFD for 4 months.\n-Quantification of the western blots would be beneficial\nAnswer: Thank you very much for this valuable suggestion. In response, we have\nperformed densitometric quantification of the relevant Western blots and included the\nquantified data in the revised figures and figure legends. These additions strengthen\nthe conclusions drawn from the immunoblotting results and provide clearer\ncomparisons between experimental groups.\n-Figure 3g-i has the EE for the females corrected by bodyweight. This should be\nchanged to the data without bodyweight correction for accurate representation\nlike the male data in the first part of Figure 3.\n(https://pubmed.ncbi.nlm.nih.gov/40993210/)\nAnswer: Thank you for this suggestion and for sharing the consensus guide. We have\nnow re-analysed the data using the same CalR method as in Figure 3, and have\nreplaced the original panels (Fig.3f,h, and i).\nFig. 3g, Indirect calorimetry study in female mice fed HFD for two weeks. Hourly energy\nexpenditure (EE) in female Gpr146⁺/⁺ and Gpr146⁻/⁻ mice was recorded over 60 hours (n=8 mice\nper group). 3h, Mean hourly energy expenditure (kcal/hour) per mouse during total, dark, and\nlight phases. * p< 0.05, ** p< 0.01\n-Following up on the EE, I suppose the adipose KO mice in indirect calorimetry,\npaired with the cell data on isolated adipocytes, would suggest the EE is not\narising from the fat tissue?\nAnswer: We thank the reviewer for the important follow-up question. The combined\ndata from our indirect calorimetry studies of adipose KO and Seahorse assays indeed\nsuggest that the increased energy expenditure (EE) observed in whole-body Gpr146\nknockout mice, particularly in males, is unlikely to originate solely from white\nadipose tissue.\nHowever, the lack of a measurable EE increase in adipose-specific Gpr146 knockout\nmice does not fully exclude an adipose contribution to the elevated EE observed in\nwhole-body knockouts. Two factors may help explain this discrepancy. First, Adipoq-\nCre-mediated deletion of GPR146 is attenuated in visceral fat depots under prolonged\nHFD feeding. This partial depletion likely contributes to the more modest reductions in\nbody weight and adiposity observed in adipose-specific knockouts compared to global\nknockouts. Second, our data suggest that GPR146 regulates both preadipocyte\ndifferentiation and lipolysis in mature adipocytes. Since Adipoq-Cre is active mainly in\nmature adipocytes, the model may not capture the full functional repertoire of GPR146\nin adipose tissue. As a result, the partial phenotypic effects in adipose-specific\nknockouts may be insufficient to elicit a detectable increase in energy expenditure by\nindirect calorimetry.\nOur results also reveal a sex-specific divergence in thermogenic responses. In female\nGpr146-/- mice, we observed a robust (>15-fold) induction of Ucp1 in eWAT,\naccompanied by upregulation of Pgc1α and Ppara, indicating potential activation of\nUCP1-mediated thermogenesis. In contrast, male knockouts showed no transcriptional\ninduction of Ucp1 or other classical thermogenic markers, and neither creatine nor\ncalcium futile cycling pathways appeared upregulated at the gene expression level.\nTaken together, these data suggest that while adipose tissue-resident GPR146 plays\nan important role in regulating adipogenesis and lipid mobilization, adipose tissue is\nunlikely to be the sole driver of the elevated energy expenditure- particularly in males.\nThe precise mechanisms underlying this phenotype remain unresolved and are likely\nto involve systemic factors, non-adipose tissue contributions, or inter-organ crosstalk.\nWe appreciate the reviewer raising this point, and we have honestly\nacknowledged that the detailed molecular mechanism by which GPR146\ndeficiency enhances energy expenditure remains unclear in male mice and\nrequires further investigation."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I appreciate the authors rigorous efforts in addressing my comments, I have no further concerns."}, "author_response": ""}]

Decision letter