{"paper_id":"d0337d90-c10a-4c31-932e-32124bdd9faa","body_text":"Serotonin drives selective hepatic insulin resistance via HTR2A and HTR2B | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Letter Serotonin drives selective hepatic insulin resistance via HTR2A and HTR2B Hail Kim, Jung Eun Nam, Inseon Hwang, Won Gun Choi, Wonsuk Choi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8819291/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Selective hepatic insulin resistance is characterized by preserved lipogenesis despite the failure to suppress gluconeogenesis, yet the detailed molecular mechanisms underlying this paradox remain incompletely understood. Here, we identify serotonin as an insulin independent regulator that differentially regulates hepatic lipid and glucose metabolism in insulin resistant states. Serotonin promoted hepatic lipogenesis through HTR2Amediated activation of the Ca 2+ -PI3K-AKT-mTORC1 signaling cascade, leading to SREBP1 activation independently of insulin receptor. In parallel, serotonin signaling through HTR2Bstimulates hepatic gluconeogenesis via a Ca 2+ -NO-cGMP-PKG pathway, resulting in CREB phosphorylation independently of PKA. Genetic disruption of HTR2A selectively attenuated hepatic lipogenesis, whereas deletion of HTR2B suppressed gluconeogenic gene expression and hepatic glucose production without affecting lipogenesis. Together, these findings establish serotonergic regulation as a dual, insulin independent driver of hepatic lipogenesis and gluconeogenesis under insulin resistant conditions, providing a mechanistic explanation for selective hepatic insulin resistance and identifying serotonin receptors as potential therapeutic targets for metabolic disease. Biological sciences/Cell biology/Cell signalling/Hormone receptors Biological sciences/Cell biology/Cell signalling/Insulin signalling Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Main The liver is a central organ for glucose and lipid metabolism, which is predominantly regulated by insulin 1 . Consequently, hepatic insulin signaling is closely linked to metabolic diseases such as type 2 diabetes and metabolic dysfunction–associated steatotic liver disease (MASLD) 2 – 4 . Under physiological conditions, insulin promotes lipogenesis and suppresses gluconeogenesis in hepatocytes 5 . Insulin, upon binding to insulin receptor (IR), triggers phosphorylation of downstream molecules, including AKT 6 . A major branch of AKT signaling leads to activation of mTORC1-SREBP1 axis, thereby promoting lipogenesis 7 . In parallel, another branch of AKT signaling leads to phosphorylation of FOXO1, a transcription factor that promotes hepatic gluconeogenesis 1 , 6 , 8 . Phosphorylated FOXO1 is retained in cytoplasm and loses its transcriptional activity 9 . Consistent with these mechanisms, liver specific IR knockout (LIRKO) mice exhibit reduced hepatic lipogenesis and increased gluconeogenesis because insulin fails to promote lipogenesis and suppress gluconeogenesis in the liver 8 , 10 . In contrast, under insulin resistant conditions, insulin loses its ability to suppress hepatic gluconeogenesis, whereas its effects on hepatic lipogenesis remain preserved 1 . Along with the influx of fatty acids released from adipose tissue through lipolysis, sustained de novo lipogenesis promotes hepatic lipid accumulation 8 , 11 , 12 . This paradox of selective hepatic insulin resistance has been explained by several hypotheses including: (1) divergence of the gluconeogenic and lipogenic pathways at a certain point in the insulin signaling cascade 5 , 13 ; (2) the presence of an insulin independent signaling pathway that activates SREBP1 and lipogenesis 1 , 14 , 15 ; (3) the differential zonal distribution and altered expression of IRS1 and IRS2 in the liver under diabetic conditions 16 . However, the underlying mechanisms of selective hepatic insulin resistance remain incompletely understood. Serotonin(5-hydroxytryptamine,5-HT) is a neurotransmitter with diverse biological functions in both central and peripheral tissues 17 – 19 . 5-HT is synthesized from essential amino acid tryptophan through sequential hydroxylation and decarboxylation reactions. The rate limiting enzyme, tryptophan hydroxylase (TPH) exists in two isoforms with distinct tissue distributions: TPH1 in peripheral tissues and TPH2 in central nervous systems 20 , 21 . Since 5-HT cannot cross the blood-brain-barrier, its central and peripheral actions are functionally separated and its biological actions are mediated by 5-HT receptor (HTR) 22 , 23 . Accumulating evidence implicates peripheral 5-HT as an important regulator of systemic metabolism, contributing to insulin resistance and hepatic steatosis 24 – 27 . In particular, gut derived 5-HT has been shown to act on hepatocytes through HTR2A to promote hepatic steatosis 24 . Conversely, pharmacological or genetic inhibition of HTR2A signaling ameliorates hepatic steatosis 27 . Despite these findings, the precise mechanisms by which 5-HT/HTR2A signaling intersects with insulin signaling pathways remain poorly defined. Here, we identify 5-HT signaling as an insulin independent regulator of hepatic metabolic selectivity. We demonstrate that distinct hepatic HTRs differentially regulate lipogenesis and gluconeogenesis. Activation of HTR2A promotes hepatic lipogenesis through the Ca 2+ -PI3K-AKT-mTORC1-SREBP1 pathway, whereas HTR2B signaling stimulates gluconeogenesis via the Ca 2+ -NO-cGMP-PKG-CREB axis. These findings define 5-HT signaling as an alternative, insulin independent mechanism that regulates hepatic lipid and glucose metabolism through receptor specific pathways and provides a mechanistic explanation for the long-standing paradox of preserved lipogenesis in the insulin resistant liver. To determine whether 5-HT signaling through HTR2A intersects with insulin signaling in hepatocytes, we first compared SREBP1 expression and AKT phosphorylation between wild-type (WT) and hepatocyte-specific Htr2a knockout ( Htr2a LKO: Alb-Cre; Htr2a f/f ) mice. Consistent with our previous finding that high fat diet (HFD) increased hepatic Srebp1c mRNA expression 24 , HFD robustly increased both precursor (pSREBP1) and nuclear (nSREBP1) forms of SREBP1 in WT livers, and this induction was abolished in Htr2a LKO livers (Fig. 1 a, Extended Data Fig. 1 a). In parallel, AKT phosphorylation was increased in the liver of HFD fed WT mice but not in Htr2a LKO mice. To directly test the effect of 5-HT, we injected 5-HT into the portal vein and examined AKT and SREBP1 activation. 5-HT increased both pSREBP1 and nSREBP1, as well as AKT phosphorylation in WT livers (Fig. 1 b, Extended Data Fig. 1 b). These 5-HT dependent responses were abolished in Htr2a LKO livers (Fig. 1 c, Extended Data Fig. 1 c) but were preserved in Htr2b LKO (Alb-Cre; Htr2b f/f ) livers (Extended Data Fig. 1 d,e), indicating that 5-HT activates hepatic AKT-SREBP1 axis through HTR2A. Next, we examined the signaling cascade in AML-12 hepatocytes. Pharmacological inhibition of AKT with MK2206 abrogated 5-HT induced SREBP1 activation and AKT phosphorylation (Fig. 1 d). 5-HT also increased phosphorylation of S6K, a downstream effector of AKT to transmit insulin signaling to SREBP1 via mTORC1 28 (Extended Data Fig. 1 f). Inhibition of mTORC1 with rapamycin abolished 5-HT induced SREBP1 activation (Extended Data Fig. 1 g). These data indicate that 5-HT/HTR2A activates SREBP1 via the AKT–mTORC1–S6K cascade in AML-12 cells 5 , 7 , 29 . To identify the upstream molecules of AKT activation in 5-HT/HTR2A signaling, we applied pathway specific inhibitors in AML-12 cells. The PI3K inhibitor wortmannin suppressed 5-HT induced AKT phosphorylation (Fig. 1 e). In contrast, IR antagonist S961 failed to inhibit 5-HT induced AKT phosphorylation, despite completely inhibiting insulin induced AKT phosphorylation (Fig. 1 f, Extended Data Fig. 1 h). These findings indicate that 5-HT/HTR2A signaling converges with insulin signaling at the level of PI3K, independently of IR. We then compared the relative potency of 5-HT and insulin for AKT phosphorylation in vivo using LIRKO (Alb-Cre; Insr f/f ) mice. In WT mice, portal vein injection of either 5-HT or insulin induced hepatic AKT phosphorylation, but the response to 5-HT was weaker than that to insulin. Interestingly, 5-HT induced AKT phosphorylation was augmented in LIRKO livers compared with WT (Fig. 1 g, Extended Data Fig. 1 i), suggesting enhanced 5-HT/HTR2A signaling in the absence of insulin signaling. In contrast, 5-HT failed to induce AKT phosphorylation in Htr2a;Insr double knockout ( Htr2a;Insr LKO) mice (Fig. 1 g, Extended Data Fig. 1 i). Collectively, these results demonstrate that 5-HT activates hepatic PI3K-AKT-SREBP1 axis through HTR2A independently of IR. HTR2A is a Gq-coupled receptor that activates phospholipase C, generating inositol 1,4,5-triphosphate (IP3) and diacylglycerol 30 . IP3 triggers Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺-bound calmodulin can activate PI3K 31 . Consistent with this model, blocking IP3 receptor signaling with 2-APB or chelating intracellular Ca²⁺ with BAPTA-AM reduced 5-HT induced AKT phosphorylation in AML-12 cells (Fig. 1 h,i). Inhibition of calmodulin with chlorpromazine (CPZ) also suppressed 5-HTinduced AKT phosphorylation (Fig. 1 j). These findings suggest that 5-HT/HTR2A signaling elevates intracellular Ca²⁺, enabling Ca²⁺/calmodulin dependent PI3K activation, AKT phosphorylation, and downstream SREBP1 activation (Fig. 1 k). To delineate the relative contributions of 5-HT and insulin signaling to hepatic lipogenesis under insulin resistant conditions, we examined the effects of 5-HT in the absence of HTR2A and/or IR in mice fed HFD. Hepatocyte specific gene deletion was achieved by AAV8-albumin-Cre in Htr2a f/f (AAV-2A LKO), Insr f/f (AAV-IR LKO), and Htr2a f/f ; Insr f/f (AAV-DR LKO) mice. One week after AAV8 injection, the mice were fed HFD for 8 weeks to induce hepatic steatosis and systemic insulin resistance. Efficient gene deletion was confirmed by hepatic mRNA and protein analyses (Fig. 2 a, Extended Data Fig. 2 a,b). In this HFD induced insulin resistant state, both SREBP1 expression and AKT phosphorylation were reduced in AAV-2A LKO and AAV-DR LKO livers compared with WT controls, whereas no significant changes were observed in AAV-IR LKO livers (Fig. 2 a,b). Concordantly, histological analysis using H&E and Oil Red O staining revealed marked reduction in lipid accumulation in AAV-2A LKO and AAV-DR LKO livers, accompanied by decrease in hepatic triglyceride content (Fig. 2 c, Extended Data Fig. 2 c). Body weight and plasma insulin levels were comparable among experimental groups (Extended Data Fig. 2 d,e), excluding differences in systemic metabolic status as confounding factors. Taken together, these results demonstrate that the enhanced hepatic lipogenesis and lipid accumulation under HFD induced insulin resistance are driven primarily by 5-HT/HTR2A signaling rather than insulin signaling. Impaired suppression of hepatic gluconeogenesis is another defining feature of selective hepatic insulin resistance and has been attributed, at least in part, to reduced AKT mediated phosphorylation of FOXO1, resulting in sustained transcription of gluconeogenic genes 1 . To assess the contribution of 5-HT to increased hepatic gluconeogenesis during insulin resistance, we examined FOXO1 phosphorylation in our models. Interestingly, FOXO1 phosphorylation was preserved in HFD fed WT mice and was instead reduced in HFD fed Htr2a LKO mice (Extended Data Fig. 3 a), indicating that 5-HT/HTR2A signaling contributes to the maintenance of AKT mediated FOXO1 phosphorylation under insulin resistant conditions. These findings argue against FOXO1 de-repression as the primary driver of gluconeogenesis in insulin resistance and suggest the existence of a FOXO1 independent mechanism that promotes hepatic gluconeogenesis. CREB is a transcription factor that promotes gluconeogenic gene expression during fasting 32 . Intriguingly, HFD increased CREB phosphorylation in both WT and Htr2a LKO mice (Fig. 3 a, Extended Data Fig. 3 b), implicating CREB as a potential mediator for increased gluconeogenesis independent of FOXO1. Consistent with this notion, direct portal injection of 5-HT increased CREB phosphorylation in WT and Htr2a LKO livers but not in Htr2b LKO livers (Fig. 3 b,c, Extended Data Fig. 3 c-f), demonstrating that 5-HT activates CREB via HTR2B. We next sought to identify signaling pathway linking HTR2B activation to CREB phosphorylation. Under physiological conditions, glucagon induces CREB phosphorylation through the adenylyl cyclase–cAMP–PKA pathway 32 . However, pharmacological inhibition of PKA with KT-5720 or adenylyl cyclase with KH-7 did not attenuate 5-HT induced CREB phosphorylation in AML-12 cells suggesting that 5-HT induces CREB phosphorylation through a PKA independent mechanism (Fig. 3 d,e). Given that 5-HT/HTR2B signaling in adipose tissue activates PKG rather than PKA 26 , we examined nitric oxide (NO)-cGMP-PKG pathway in AML-12 cells. Inhibition of PKG (KT-5823), guanylyl cyclase (ODQ), or NO synthase (ADMA) reduced 5-HT induced CREB phosphorylation (Fig. 3 f-h). In addition, inhibition of intracellular Ca²⁺ signaling with 2-APB or BAPTA-AM reduced CREB phosphorylation (Fig. 3 i,j). These results indicate that 5-HT/HTR2B signaling increases intracellular Ca²⁺, activates NO synthase 33 , leading to NO production that stimulates guanylyl cyclase-cGMP-PKG pathway 26 , 34 to promote CREB phosphorylation (Fig. 3 k). To evaluate the physiological relevance of hepatic 5-HT/HTR2B signaling in vivo , we examined Htr2b LKO mice after 8weeks of HFD feeding. Body weight and hepatic triglyceride content were comparable between WT and Htr2b LKO mice, although baseline glucose tolerance was slightly improved at 15 minutes in Htr2b LKO mice (Extended Data Fig. 4 a-c). In contrast, after 8 weeks of HFD feeding, glucose tolerance was markedly improved in Htr2b LKO mice without a corresponding improvement in systemic insulin sensitivity (Fig. 4 a,b). In addition, pyruvate tolerance tests revealed reduced hepatic glucose production in Htr2b LKO mice (Fig. 4 c, Extended Data Fig. 4 d), suggesting that improved glucose tolerance is attributed to reduced gluconeogenesis. At molecular level, HFD increased hepatic CREB phosphorylation in WT mice, which was abolished in Htr2b LKO mice, whereas FOXO1 phosphorylation remained unchanged (Fig. 4 d). Correspondingly, hepatic mRNA expression of gluconeogenic genes including G6pc , Pck1 , and Fbp1 , was decreased in HFD fed Htr2b LKO mice, whereas lipogenic genes ( Srebf1, Acly, Acaca, Fasn, Scd1 ) remained unchanged (Fig. 4 e). Together, these results demonstrate that 5-HT/HTR2B signaling promotes hepatic gluconeogenesis through PKG–CREB signaling axis, while hepatic lipogenesis under HFD induced insulin resistance remains selectively driven by 5-HT/HTR2A signaling. In this study, we identify 5-HT as a key determinant of selective hepatic insulin resistance by demonstrating that it regulates hepatic lipogenesis and gluconeogenesis under insulin resistance, independently of insulin signaling. We show that hepatic HTR2A activation promoted lipogenesis via the AKT-mTORC1-S6K pathway, leading to SREBP1 activation and induction of lipogenic gene expression. In parallel, hepatic HTR2B activation promoted gluconeogenesis through the guanylyl cyclase-cGMP-PKG signaling pathway, resulting in CREB phosphorylation and induction of gluconeogenic gene expression. These findings establish 5-HT signaling as an insulin independent alternative pathway that regulates hepatic metabolic control in insulin resistant states, providing a mechanistic explanation for the long-standing metabolic paradox of preserved lipogenesis despite impaired insulin action in the liver. Insulin is the principal regulator of hepatic lipid and glucose metabolism, transmitting signals through the IR-IRS1/IRS2-PI3K-AKT axis to multiple downstream branches, including FOXO1 and mTORC1 1,6,9,28 . Genetic dissection of these signaling components has demonstrated that failure of AKT to phosphorylate and inactivate FOXO1 leads to increased gluconeogenesis in insulin resistance 5 , 6 , 8 , 10 , 16 . Likewise, impaired insulin induced AKT phosphorylation reduces SREBP1 activation and lipogenesis. Accordingly, mice lacking these signaling components consistently display increased hepatic glucose production and reduced lipogenesis, a phenotype characteristic of ‘total hepatic insulin resistance’. However, these findings are inconsistent with the metabolic features observed in patients with type 2 diabetes and in insulin resistant animal models induced by HFD feeding or genetic defects, such as ob/ob and db/db mice 1 , 8 , 13 , 35 , 36 . In these models, hepatic gluconeogenesis and lipogenesis are concomitantly increased, a paradoxical phenomenon termed ‘selective hepatic insulin resistance’. Although this paradox has been attributed to differential impairment of insulin signaling 1 , 13 , this concept cannot fully explain the persistent lipogenesis despite diminished insulin responsiveness 8 , 14 . Unlike models of total insulin resistance, models of type 2 diabetes show elevated basal AKT phosphorylation despite reduced responsiveness to exogenous insulin 8 , 37 , suggesting the presence of additional signaling inputs that sustain both lipogenesis and gluconeogenesis. Previously, we demonstrated that liver-specific deletion of Htr2a reduced lipogenic gene expression and ameliorated hepatic steatosis in HFD fed mice, suggesting 5-HT/HTR2A as such an additional signal. Here, we extend these observations by defining the molecular mechanisms through which 5-HT independently regulates hepatic lipogenesis and gluconeogenesis. We present that 5-HT/HTR2A activated the PI3K-AKT-mTORC1-SREBP1 pathway through Ca 2+ -calmodulin dependent mechanism that occurred independently of the IR. In parallel, we identify a distinct role for 5-HT/HTR2B in regulating hepatic gluconeogenesis. Upon binding to HTR2B, 5-HT initiated a Ca 2+ -NO-cGMP-PKG signaling cascade that led to CREB phosphorylation. Deletion of Htr2b in hepatocytes decreased gluconeogenic gene expression and hepatic glucose production under HFD feeding. Importantly, this pathway operated independently of insulin mediated FOXO1 regulation 6 or glucagon mediated PKA signaling 38 , 39 . Our findings highlight an important role of CREB in driving hepatic gluconeogenesis in insulin resistant state and are consistent with previous studies, demonstrating that CREB activity contributes to fasting hyperglycemia in db/db diabetic mice in which CREB inhibition attenuated hepatic glucose production and normalized plasma glucose levels 10 , 32 , 38 , 40 . Our data place CREB downstream of 5-HT/HTR2B signaling, providing a mechanistic link between elevated portal 5-HT and dysregulated hepatic glucose production. Collectively, these findings identified 5-HT as a dual regulator of hepatic lipid and glucose metabolism under insulin resistant conditions, acting through HTR2A and HTR2B to promote lipogenesis and gluconeogenesis, respectively (Fig. 4 f). Given that HFD feeding increases 5-HT production in the gut and Htr2a expression in the liver 24 , serotonergic signaling likely compensates for impaired insulin action and becomes a dominant driver of hepatic lipogenesis and gluconeogenesis in HFD fed mice. Therefore, we propose a new model to explain ‘selective hepatic insulin resistance’, in which non-insulin serotonergic signaling becomes pathologically dominant when insulin action is diminished. This suggests that HTR2A and HTR2B could be promising therapeutic targets for MASLD, type 2 diabetes, and related metabolic diseases. Methods Animals The male mice were bred and maintained in specific pathogen-free barrier facilities. The facilities were kept under iso-temperature and iso-humidity conditions with a 12-hour light/dark cycle. The mice had ad libitum access to diet and water. The animal experiments were approved by the Institutional Animal Care and Use Committee at the Korea Advanced Institute of Science and Technology. All experiments were performed under guidelines and regulations. To induce diet induced insulin resistance model, 12, 13-week-old mice were adjusted to high-fat diet (HFD; 60% kcal fat, D12492, Research Diets) for 8 weeks. For normal chow diet (NCD) group, the mice were provided standard chow diet during same period to compare the effect of HFD induced insulin resistance. 12-week-old floxed mice ( Htr2a f/f , Insr f/f and Htr2a f/f ; Insr f/f ) were used to inject AAV which has Alb-Cre plasmid (CV17208-AV8, Charles River) at 3ⅹ10 12 GC/kg via tail vein to express hepatocyte specific Cre. After one week from injection, the mice were adjusted to HFD for 8 weeks. At the end of the experiments, mice were anesthetized ad libitum , after which blood samples were collected for subsequent analyses. Following blood collection, mice were euthanized and tissues were rapidly harvested. The collected tissues were either frozen and stored at -80℃ or immediately processed for downstream experimental procedures. Cell culture AML-12 cell line (CRL-2254, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, SH30243.01, Hyclone) supplemented with 100 µg/ml penicillin/streptomycin (15140-122, Gibco) and 10% Fetal Bovine Serum (16000-044, Gibco) at 37 ℃ in a humidified 5% CO 2 atmosphere. Cell culture media was changed every 48 hours and subcultured at 70–80% confluence using 0.25% trypsin-EDTA solution (25200-056, Gibco). Cells at 80–90% confluence were used in experiments. Cells were treated with 100nM 5-HT (H9523, sigma), MK2206 (HY-10358, MedCamExpress), Wortmannin (681675, sigma), S961 (051–86, Phoenix pharm), 2-APB (100065, sigma), BAPTA-AM (A1076, sigma), Chlorpromazine (CPZ, 31679, Supelco), KH-7 (K3394, sigma), KT-5720 (420320, sigma), ODQ (495320, sigma), KT-5823 (420321, sigma) or ADMA (D4268, sigma). Cells were pretreated with the indicated inhibitors for 30 minutes before 5-HT treatment. Before experiments, cells for AKT and SREBP1 induction analysis were serum deprived for 6 hours, while those for CREB activation were serum deprived for 2 hours. Portal vein injection Mice for portal vein injection were anesthetized via intraperitoneal injection of avertin with combination of isoflurane inhalation. The peritoneum was opened by a midline incision and the portal vein was exposed. Insulin syringe (BD, 328820) with 100 µl PBS, 20 ng 5-HT in 100 µl PBS or 2 ng insulin in 100 µl PBS was carefully inserted into the portal vein. After 5 minutes, liver tissue was harvested and mice were sacrificed. Before injection, the mice designated for AKT phosphorylation analysis were fasted for 16 hours, while those for CREB phosphorylation were fasted for 4 hours. Westernblot analysis Liver tissue and fully cultured AML-12 cells were extracted by RIPA-Lysis extraction buffer (89901, Thermo scientific) containing protease inhibitor (P3100-010, GenDEPOT) and phosphatase inhibitor (P3200-010, GenDEPOT). Tissue samples in bead tubes were homogenized by Fast Prep-24 (MP Biomedicals). After protein extraction, suspensions were centrifuged at 13000 rpm at 4 ℃ for 15 minutes. To determine protein concentration, the BCA Protein Assay Kit (23227, Pierce) was performed; 10–20 µg of protein was subjected to 8–10% SDS-PAGE gels and transferred to the PVDF membranes (IPVH00010, Millipore) which are activated by methanol. Transferred membranes were incubated in the blocking solution (TBS buffer with 0.1% Tween 20) containing 5% BSA (w/v ratio) (0210370380, Mpbio) for 1 hour at room temperature. All antibodies used in this study are listed in Supplementary Table. Primary antibodies were reacted overnight at 4 ℃; secondary antibodies were reacted for 1 hour 30 minutes at room temperature. ECL substrate (WBKLS0500, Millipore) was used to amplify the detectable signals for ImageQuant 800 (cytiva, Amercham). The band intensities were quantified and represented in bar graphs. Quantitative PCR (qPCR) analysis For mRNA extraction to examine transcriptional levels, liver tissue samples in bead tube or treated cells were collected in Tri-RNA reagent (15596-018, Ambion) and homogenized by Fast Prep-24 (MP Biomedicals). And extraction protocol, which includes chloroform (67-66-3, Junsei) and iso-propyl alcohol (67-63-0, Supelco), was used. After extraction, cDNA was synthesized from 1 µg of total RNA using a High Capacity of cDNA Reverse Transcriptase Kit (4368813, Applied Biosystems, Foster Cuy, CA, USA). Quantitative real time-polymerase chain reaction (qPCR) was performed on a ViiA7 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using Fast SYBR Green Master Mix (4385612, Applied Biosystems) according to the manufacturer’s instructions. Expressional profiles were quantified according to the relative ddCt method using 36B4 as a reference gene. The sequence of primers used in this study are listed in Supplementary Table. Hematoxylin & Eosin (H&E) staining For H&E staining, harvested liver tissues were fixed in formalin and embedded in paraffin. Five-micron-thick sections were deparaffinized, rehydrated, and stained with H&E (AR173, Agilent Dako, Santa Clara, CA, USA), according to the manufacturer’s instruction. Oil red O staining For Oil red O staining, harvested liver tissues were fixed in 4% paraformaldehyde for 4 hours and dehydrated serially by 20% sucrose and 40% sucrose solution. Dehydrated tissues are frozen in Tissue Tek. O.C.T compound (4583, Sakura). The frozen liver sections were cut at 8–10 micron-thick and dry at tabletop. After washing by tap water for 5 minutes, the sections were rinsed with 60% isopropanol. Prepared Oil red O working solution, which is mixture of 0.5% Oil red O stock solution in isopropanol with distilled water (DW) at 6:4 ratio, was used to stain the rinsed section for 15 minutes. After staining, the section was rinsed with 60% isopropanol and stained with hematoxylin for 10 seconds to stain nuclei. Then, rinsed sections by DW were mounted with a medium under the cover glass. Images were acquired by bright-field microscope (Nikon eclipse Ni). Glucose/Pyruvate/Insulin tolerance test (GTT/PTT/ITT) For glucose tolerance test (GTT), the mice were fasted overnight for 16 hours and 2 g/kg D-glucose (20%, Dai han pharm) was injected in mice intraperitoneally. For glucose measurement, blood glucose levels from 0, 15, 30, 60, 90 and 120 minutes after injection were measured from tail vein using a glucometer (GlucoDr.TOP glucometer, Allmedicus). For pyruvate tolerance test (PTT) and insulin tolerance test (ITT), the mice were fasted for 6 hours and 2 g/kg-sodium pyruvate (P5280, Sigma) and 1 U/kg insulin (I9278, Sigma), respectively, was injected in mice intraperitoneally. For glucose measurement, blood glucose levels were measured from tail vein using a glucometer (GlucoDr.TOP glucometer, Allmedicus) at indicated time points after injection. Quantification of hepatic triglycerides (TG) level Liver tissues were homogenized in 5% NP-40 using Fast Prep-24 (MP Biomedicals). TGs were solubilized by two cycles of heating homogenates to 95°C for 5 minutes and then, cooling on ice. Triglyceride Reagent (T2449, sigma) or DW was added, and TGs were hydrolyzed into glycerol by incubating samples at 37°C for 30 minutes. For colorimetric assay of hydrolyzed TG levels, samples were incubated with Free Glycerol Reagent (F6428, sigma) at 37°C for 5 minutes. Differences in absorbance at 540 nm between hydrolyzed and non-hydrolyzed TGs were quantified using a glycerol standard (G7793, sigma). TG content was normalized to the protein concentration in homogenates, measured using a BCA Protein Assay Kit (23227, Pierce). Insulin ELISA Plasma insulin concentrations were measured using insulin immunoassay kit (#80-INSMSU, ALPCO) according to the manufacturer’s instructions. Briefly, whole blood was collected into EDTA-coated tubes, and centrifuged at 2,000 g for 5 minutes at 4°C to separate plasma. Plasma samples were aliquoted and stored at -80°C until analysis. Prior to assay samples were thawed on ice and briefly centrifuged to remove precipitates. All standards, controls and samples were assayed in duplicate. Quantification and statistical analysis Values from every experiment are expressed as the mean ± standard error of mean (SEM). To compare groups, the two-tailed Student’s t test or one-way analysis of variance (ANOVA) followed by post-hoc Tukey’s test were used. P values below 0.05 were considered statistically significant. The levels of significance indicated in the graphs are *; p < 0.05, **; p < 0.01, ***; p < 0.001, ****; p < 0.0001. Declarations Acknowledgements This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT, Ministry of Science and ICT) (No. RS-2025-00513814 and No. RS-2025-02214748), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. RS-2025-02262990) (to H.K.), Inha University (No. 71580 to Y.A.M.) and the National Research Foundation of Korea (NRF) (No. RS-2022-00166199 to I.H., No. RS-2023-00247558 to W.G.C, No. RS-2024-00408919 to W.C., and No. RS-2024-00453271 to W.I.C.). Author contributions I.H., J.E.N., Y.M. and H.K. conceived the study. I.H., W.G.C., W.C., W.I.C. and J.E.N. performed experiments. J.E.N., Y.M. and H.K. wrote the manuscript. H.K. and Y.M supervised the project. I.H. and W.C. edited the manuscript. Competing interests The authors declare no competing interests. References Bo, T. et al. 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Serotonin regulates pancreatic beta cell mass during pregnancy. Nat. Med. 16, 804-808 (2010). https://doi.org:10.1038/nm.2173 Choi, W. G. et al. Inhibiting serotonin signaling through HTR2B in visceral adipose tissue improves obesity-related insulin resistance. J. Clin. Invest. 131 e145331 (2021). https://doi.org/10.1172/JCI145331 Pagire, H. S. et al. Discovery of a peripheral 5HT2A antagonist as a clinical candidate for metabolic dysfunction-associated steatohepatitis. Nat. Commun. 15 , 645 (2024). https://doi.org/10.1038/s41467-024-44874-3 Yecies, J. L. et al. Akt stimulates hepatic SREBP1c and lipogenesis through parallel mTORC1-dependent and independent pathways. Cell Metab. 14, 21-32 (2011). https://doi.org:10.1016/j.cmet.2011.06.002 Bakan, I. & Laplante, M. Connecting mTORC1 signaling to SREBP-1 activation. Curr. Opin. Lipidol. 23, 226-234 (2012). https://doi.org:10.1097/MOL.0b013e328352dd03 Yabut, J. M. et al. 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Nutrient control of phosphorylation and translocation of FoxO1 in C57BL/6 and db/db mice. Int. J. Mol. Med. 18, 433-439 (2006). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable.xlsx Supplementary Table Additionalinformation.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8819291\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Letter\",\"associatedPublications\":[],\"authors\":[{\"id\":591463549,\"identity\":\"4ab6fe91-c818-4294-aa06-be189024aa10\",\"order_by\":0,\"name\":\"Hail Kim\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACAwbGxoMNBgxyEgyMDSABCElASwNIizFYywHitDAwHASqSpwB4hGlxZz9cMPBGQXb0me2H27+/IHBRnbDAQJaLHsSGw5uMLidO5snsU3iAEOaMUEtBgeAWh4AtcxjSGwDOuxwImEt5x+CtaTL8T9s/nCA4T8RWm5AHJYgLZHYAHTYAWK0AG2ZYXDbcOaMh20SZwySjWcSdlj6w4c9f27LS5xPf/yhosJOto+QFnQTSFM+CkbBKBgFowAHAABLb1NjpmJJpgAAAABJRU5ErkJggg==\",\"orcid\":\"https://orcid.org/0000-0002-6652-1349\",\"institution\":\"Korea Advanced Institute of Science and Technology (KAIST)\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Hail\",\"middleName\":\"\",\"lastName\":\"Kim\",\"suffix\":\"\"},{\"id\":591463550,\"identity\":\"16df8f68-ad08-409e-ab2a-dbb2d4553eb7\",\"order_by\":1,\"name\":\"Jung Eun Nam\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0002-7021-8406\",\"institution\":\"Korea Advanced Institute of Science and Technology (KAIST)\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jung\",\"middleName\":\"Eun\",\"lastName\":\"Nam\",\"suffix\":\"\"},{\"id\":591463551,\"identity\":\"fe5db7cd-811c-437c-bb33-8e0a1bf8206a\",\"order_by\":2,\"name\":\"Inseon Hwang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Daejeon Health Institute of Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Inseon\",\"middleName\":\"\",\"lastName\":\"Hwang\",\"suffix\":\"\"},{\"id\":591463552,\"identity\":\"5c70fca5-1c16-44e0-bfa4-96d77fdf0584\",\"order_by\":3,\"name\":\"Won Gun Choi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"The Catholic University of Korea\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Won\",\"middleName\":\"Gun\",\"lastName\":\"Choi\",\"suffix\":\"\"},{\"id\":591463553,\"identity\":\"dc49cc88-5937-4c6d-9dd1-6c337d696da2\",\"order_by\":4,\"name\":\"Wonsuk Choi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Korea Advanced Institute of Science and Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Wonsuk\",\"middleName\":\"\",\"lastName\":\"Choi\",\"suffix\":\"\"},{\"id\":591463554,\"identity\":\"7f965fab-2a0d-4c0b-821f-aa937a38e7ca\",\"order_by\":5,\"name\":\"Won-Il Choi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jeonbuk National University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Won-Il\",\"middleName\":\"\",\"lastName\":\"Choi\",\"suffix\":\"\"},{\"id\":591463555,\"identity\":\"1f02f13b-21c2-4829-ab57-4e71b181e9ff\",\"order_by\":6,\"name\":\"Young-Ah Moon\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Inha University College of Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Young-Ah\",\"middleName\":\"\",\"lastName\":\"Moon\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-02-08 05:25:49\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-8819291/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-8819291/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":103881732,\"identity\":\"a962840e-3653-4269-b182-ec84f29fbfcf\",\"added_by\":\"auto\",\"created_at\":\"2026-03-04 05:40:51\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2978455,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e5-HT induces the AKT-SREBP1 axisvia HTR2A, independently of insulin signaling.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea, \\u003c/strong\\u003eTwelve-week-old male WT and \\u003cem\\u003eHtr2a \\u003c/em\\u003eLKO mice were fed an NCD or an HFD for 8 weeks. Whole cell lysates were prepared from the liver and subjected to immunoblot analysis for precursor (p) and nuclear (n) forms of SREBP1, phosphorylated AKT (pAKT) and total AKT. GAPDH was used as a loading control. \\u003cstrong\\u003eb-c, \\u003c/strong\\u003eWT (B) and \\u003cem\\u003eHtr2a \\u003c/em\\u003eLKO (C) mice were fasted for 16 hours and injected with20 ng/ 100 μl of 5-HT via the portal vein. 5 minutes after injection, the liver was collected, whole-cell lysates were prepared, and immunoblot analysis was performed for SREBP1 and AKT. \\u003cstrong\\u003ed-f, \\u003c/strong\\u003eAML-12 cells were treated with the indicated amount of MK2206 (\\u003cstrong\\u003ed\\u003c/strong\\u003e), Wortmannin (nM) (\\u003cstrong\\u003ee\\u003c/strong\\u003e), or S961 (\\u003cstrong\\u003ef\\u003c/strong\\u003e) in the presence or absence of 100 nM 5-HT for 5 min. Whole-cell lysates were prepared and subjected to immunoblot analysis for SREBP1 or AKT. Band intensities were quantified and normalized to GAPDH. \\u003cstrong\\u003eg, \\u003c/strong\\u003eTwelve-week-old male WT, hepatocyte specific IR knockout (LIRKO), and \\u003cem\\u003eHtr2a\\u003c/em\\u003e;IR double knockout (\\u003cem\\u003eHtr2a;Insr\\u003c/em\\u003eLKO) micewere fasted for 16 hours and injected with 2 ng/ 100 μl of insulin or 20 ng/ 100 μlof 5-HT via the portal vein. Five minutes after injection, the liver was collected, and whole-cell lysates were subjected to immunoblot analysis for IR and AKT. \\u003cstrong\\u003eh-j,\\u003c/strong\\u003e AML-12 cells were treated with the indicated amount of 2-APB (\\u003cstrong\\u003eh\\u003c/strong\\u003e), BAPTA-AM (μM) (\\u003cstrong\\u003ei\\u003c/strong\\u003e), or CPZ (\\u003cstrong\\u003ej\\u003c/strong\\u003e) in the presence or absence of 100 nM 5-HT or 10 nM insulin for 5 min. Whole-cell lysates were prepared, and AKT phosphorylation was determined by immunoblot analysis. \\u003cstrong\\u003ek\\u003c/strong\\u003e, Schematic representation of the proposed model showing the induction of SREBP1 by 5-HT through HTR2A. Data are presented as the mean ± S.E. (n=4). Statistical significance was determined by unpaired two-tailed \\u003cem\\u003et\\u003c/em\\u003e-test for not-treated and 5-HT treated sample and one-way ANOVA followed by Tukey’s multiple comparisons test for inhibitor treated samples, *p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8819291/v1/66e560d2c824323b5c1645c8.png\"},{\"id\":103881744,\"identity\":\"668091a0-16b1-4218-ab4e-5c65de7fdb47\",\"added_by\":\"auto\",\"created_at\":\"2026-03-04 05:40:52\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":5677861,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e5-HT drives hepatic steatosis via insulinindependent activation of the AKT-SREBP1 pathway\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea, \\u003c/strong\\u003eTwelve-week-old male WT, \\u003cem\\u003eHtr2a\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eInsr\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eHtr2a\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e;Insr\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e mice were injected with AAV-albumin-Cre virus. One week after injection, the mice were fed an HFD for 8 weeks.\\u0026nbsp; Immunoblot analyses of SREBP1, phosphorylated AKT and IR were performed using liver whole-cell lysates. \\u003cstrong\\u003eb, \\u003c/strong\\u003e\\u003cem\\u003eSrebp1c\\u003c/em\\u003e mRNA expression levels were quantified by qPCR. \\u003cstrong\\u003ec, \\u003c/strong\\u003eRepresentative images of hematoxylin and eosin (H\\u0026amp;E)- and Oil red O-stained liver section from the mice. Scale bars, 100μm.Data are presented as the mean ± S.E. (n=7). Statistical significance was determined by unpaired t test, \\u003cem\\u003et\\u003c/em\\u003e-test, *\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.05, **\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.01, ***\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.001, ****\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8819291/v1/b5b9427c97451fb2459eb7dd.png\"},{\"id\":103881767,\"identity\":\"1995ad57-374e-4f83-aaa6-cef7866276a0\",\"added_by\":\"auto\",\"created_at\":\"2026-03-04 05:40:53\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2162600,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eHTR2Bmediated hepatic gluconeogenesis inducedby5-HTviaCREB\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea, \\u003c/strong\\u003eLiver extracts were prepared from the mice described in Fig. 1a, and immunoblot analyses were performed for CREB. \\u003cstrong\\u003eb-c, \\u003c/strong\\u003eTwelve-week-old male WT, \\u003cem\\u003eHtr2a\\u003c/em\\u003e LKO, and \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO mice were fasted for 4 hours and injected with 20 ng/ 100 μl of 5-HT via the portal vein. 5 minutes after injection, the livers were harvested, whole cell lysates were prepared, and immunoblot analyses were performed to determine CREB phosphorylation. \\u003cstrong\\u003ed-j,\\u003c/strong\\u003e AML-12 cells were treated with 100nM 5-HT in the presence or absence of the indicated inhibitors: KT-5720 (\\u003cstrong\\u003ed\\u003c/strong\\u003e), KH-7 (\\u003cstrong\\u003ee\\u003c/strong\\u003e), KT-5823 (\\u003cstrong\\u003ef\\u003c/strong\\u003e), ODQ (\\u003cstrong\\u003eg\\u003c/strong\\u003e), ADMA (\\u003cstrong\\u003eh\\u003c/strong\\u003e), 2-APB (\\u003cstrong\\u003ei\\u003c/strong\\u003e), or BAPTA-AM (μM) (\\u003cstrong\\u003ej\\u003c/strong\\u003e). Immunoblot analyses were performed using whole cell lysates to assess CREB phosphorylation. \\u003cstrong\\u003ek\\u003c/strong\\u003e, Schematic representation of the proposed pathway for HTR2B mediated CREB activation in hepatocytes. Data are presented as mean ± SEM. Statistical significance was determined by unpaired \\u003cem\\u003et\\u003c/em\\u003e-test for not-treated and 5-HT treated sample and one-way ANOVA followed by Tukey’s multiple comparisons test for inhibitor treated samples, *\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.05, **\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.01, ***\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.001, ****\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8819291/v1/ad908ffaaef8fa73dfb1b937.png\"},{\"id\":103881764,\"identity\":\"04a68574-b16f-4a0c-aed7-001aecf69108\",\"added_by\":\"auto\",\"created_at\":\"2026-03-04 05:40:53\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3236325,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eGluconeogenic effect of HTR2B induced by 5-HT\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ea, \\u003c/strong\\u003eAfter8 weeks of HFD feeding, WT and \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO mice were fasted for 16 hours, and a glucose tolerance test (GTT) was performed. \\u003cstrong\\u003eb-c, \\u003c/strong\\u003eAfter7 weeks of HFD feeding, WT and \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO mice were fasted for 6 hours, after which an insulin tolerance test (ITT) (\\u003cstrong\\u003eb\\u003c/strong\\u003e) and a pyruvate tolerance test (PTT) (\\u003cstrong\\u003ec\\u003c/strong\\u003e) were performed on different days. \\u003cstrong\\u003ed\\u003c/strong\\u003e, Twelve-week-old male WT and \\u003cem\\u003eHtr2b\\u003c/em\\u003eLKO were fed an NCD or an HFD for 8 weeks. Whole cell lysates were prepared from the liver and immunoblot analyses were performed for FOXO1andCREB. \\u003cstrong\\u003ee\\u003c/strong\\u003e, The mRNA expression of gluconeogenic genes,\\u003cem\\u003eG6pc, Pck1,\\u003c/em\\u003eand\\u003cem\\u003e Fbp1\\u003c/em\\u003eandlipogenic genes,\\u003cem\\u003eSrebp1c, Acly, Acaca, Fasn, \\u003c/em\\u003eand\\u003cem\\u003eScd1\\u003c/em\\u003e,were determined by qPCR. \\u003cstrong\\u003ef\\u003c/strong\\u003e, Schematic illustration of this study. Data are presented as mean ± SEM. Statistical significance was determined by unpaired \\u003cem\\u003et\\u003c/em\\u003e-test, *\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.05, **\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.01,***\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.001, ****\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8819291/v1/f37a737e3cf08d5aaa638ea4.png\"},{\"id\":103881770,\"identity\":\"67623ab5-ec21-4cc5-8be4-7ed820e755dd\",\"added_by\":\"auto\",\"created_at\":\"2026-03-04 05:41:06\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":14794165,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8819291/v1/3b9d164e-91e8-4432-9564-bd77b131be9a.pdf\"},{\"id\":103881734,\"identity\":\"07dace00-4098-4519-b960-7947ff5f3c1c\",\"added_by\":\"auto\",\"created_at\":\"2026-03-04 05:40:51\",\"extension\":\"xlsx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":12536,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplementary Table\",\"description\":\"\",\"filename\":\"SupplementaryTable.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8819291/v1/44c30fa87d081e770b402158.xlsx\"},{\"id\":103881731,\"identity\":\"fc79cb25-7bb3-4267-ad3b-e0d67ac62be1\",\"added_by\":\"auto\",\"created_at\":\"2026-03-04 05:40:50\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1709401,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Additionalinformation.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8819291/v1/a472693bbdde61f37a808573.docx\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"Serotonin drives selective hepatic insulin resistance via HTR2A and HTR2B\",\"fulltext\":[{\"header\":\"Main\",\"content\":\"\\u003cp\\u003eThe liver is a central organ for glucose and lipid metabolism, which is predominantly regulated by insulin\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. Consequently, hepatic insulin signaling is closely linked to metabolic diseases such as type 2 diabetes and metabolic dysfunction\\u0026ndash;associated steatotic liver disease (MASLD)\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR3\\\" citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. Under physiological conditions, insulin promotes lipogenesis and suppresses gluconeogenesis in hepatocytes\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. Insulin, upon binding to insulin receptor (IR), triggers phosphorylation of downstream molecules, including AKT\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. A major branch of AKT signaling leads to activation of mTORC1-SREBP1 axis, thereby promoting lipogenesis\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e. In parallel, another branch of AKT signaling leads to phosphorylation of FOXO1, a transcription factor that promotes hepatic gluconeogenesis\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. Phosphorylated FOXO1 is retained in cytoplasm and loses its transcriptional activity\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e. Consistent with these mechanisms, liver specific IR knockout (LIRKO) mice exhibit reduced hepatic lipogenesis and increased gluconeogenesis because insulin fails to promote lipogenesis and suppress gluconeogenesis in the liver\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eIn contrast, under insulin resistant conditions, insulin loses its ability to suppress hepatic gluconeogenesis, whereas its effects on hepatic lipogenesis remain preserved\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. Along with the influx of fatty acids released from adipose tissue through lipolysis, sustained \\u003cem\\u003ede novo\\u003c/em\\u003e lipogenesis promotes hepatic lipid accumulation\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e. This paradox of selective hepatic insulin resistance has been explained by several hypotheses including: (1) divergence of the gluconeogenic and lipogenic pathways at a certain point in the insulin signaling cascade\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e; (2) the presence of an insulin independent signaling pathway that activates SREBP1 and lipogenesis\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e; (3) the differential zonal distribution and altered expression of IRS1 and IRS2 in the liver under diabetic conditions\\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e. However, the underlying mechanisms of selective hepatic insulin resistance remain incompletely understood.\\u003c/p\\u003e \\u003cp\\u003eSerotonin(5-hydroxytryptamine,5-HT) is a neurotransmitter with diverse biological functions in both central and peripheral tissues\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR18\\\" citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e. 5-HT is synthesized from essential amino acid tryptophan through sequential hydroxylation and decarboxylation reactions. The rate limiting enzyme, tryptophan hydroxylase (TPH) exists in two isoforms with distinct tissue distributions: TPH1 in peripheral tissues and TPH2 in central nervous systems\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. Since 5-HT cannot cross the blood-brain-barrier, its central and peripheral actions are functionally separated and its biological actions are mediated by 5-HT receptor (HTR)\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eAccumulating evidence implicates peripheral 5-HT as an important regulator of systemic metabolism, contributing to insulin resistance and hepatic steatosis\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR25 CR26\\\" citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e. In particular, gut derived 5-HT has been shown to act on hepatocytes through HTR2A to promote hepatic steatosis\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. Conversely, pharmacological or genetic inhibition of HTR2A signaling ameliorates hepatic steatosis\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e. Despite these findings, the precise mechanisms by which 5-HT/HTR2A signaling intersects with insulin signaling pathways remain poorly defined.\\u003c/p\\u003e \\u003cp\\u003eHere, we identify 5-HT signaling as an insulin independent regulator of hepatic metabolic selectivity. We demonstrate that distinct hepatic HTRs differentially regulate lipogenesis and gluconeogenesis. Activation of HTR2A promotes hepatic lipogenesis through the Ca\\u003csup\\u003e2+\\u003c/sup\\u003e-PI3K-AKT-mTORC1-SREBP1 pathway, whereas HTR2B signaling stimulates gluconeogenesis via the Ca\\u003csup\\u003e2+\\u003c/sup\\u003e-NO-cGMP-PKG-CREB axis. These findings define 5-HT signaling as an alternative, insulin independent mechanism that regulates hepatic lipid and glucose metabolism through receptor specific pathways and provides a mechanistic explanation for the long-standing paradox of preserved lipogenesis in the insulin resistant liver.\\u003c/p\\u003e \\u003cp\\u003eTo determine whether 5-HT signaling through HTR2A intersects with insulin signaling in hepatocytes, we first compared SREBP1 expression and AKT phosphorylation between wild-type (WT) and hepatocyte-specific \\u003cem\\u003eHtr2a\\u003c/em\\u003e knockout (\\u003cem\\u003eHtr2a\\u003c/em\\u003e LKO: Alb-Cre; \\u003cem\\u003eHtr2a\\u003c/em\\u003e\\u003csup\\u003ef/f\\u003c/sup\\u003e) mice. Consistent with our previous finding that high fat diet (HFD) increased hepatic \\u003cem\\u003eSrebp1c\\u003c/em\\u003e mRNA expression\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e, HFD robustly increased both precursor (pSREBP1) and nuclear (nSREBP1) forms of SREBP1 in WT livers, and this induction was abolished in \\u003cem\\u003eHtr2a\\u003c/em\\u003e LKO livers (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea). In parallel, AKT phosphorylation was increased in the liver of HFD fed WT mice but not in \\u003cem\\u003eHtr2a\\u003c/em\\u003e LKO mice.\\u003c/p\\u003e \\u003cp\\u003eTo directly test the effect of 5-HT, we injected 5-HT into the portal vein and examined AKT and SREBP1 activation. 5-HT increased both pSREBP1 and nSREBP1, as well as AKT phosphorylation in WT livers (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb). These 5-HT dependent responses were abolished in \\u003cem\\u003eHtr2a\\u003c/em\\u003e LKO livers (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec) but were preserved in \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO (Alb-Cre; \\u003cem\\u003eHtr2b\\u003c/em\\u003e\\u003csup\\u003ef/f\\u003c/sup\\u003e) livers (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed,e), indicating that 5-HT activates hepatic AKT-SREBP1 axis through HTR2A.\\u003c/p\\u003e \\u003cp\\u003eNext, we examined the signaling cascade in AML-12 hepatocytes. Pharmacological inhibition of AKT with MK2206 abrogated 5-HT induced SREBP1 activation and AKT phosphorylation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed). 5-HT also increased phosphorylation of S6K, a downstream effector of AKT to transmit insulin signaling to SREBP1 via mTORC1\\u003csup\\u003e28\\u003c/sup\\u003e (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef). Inhibition of mTORC1 with rapamycin abolished 5-HT induced SREBP1 activation (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eg). These data indicate that 5-HT/HTR2A activates SREBP1 via the AKT\\u0026ndash;mTORC1\\u0026ndash;S6K cascade in AML-12 cells\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eTo identify the upstream molecules of AKT activation in 5-HT/HTR2A signaling, we applied pathway specific inhibitors in AML-12 cells. The PI3K inhibitor wortmannin suppressed 5-HT induced AKT phosphorylation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ee). In contrast, IR antagonist S961 failed to inhibit 5-HT induced AKT phosphorylation, despite completely inhibiting insulin induced AKT phosphorylation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eh). These findings indicate that 5-HT/HTR2A signaling converges with insulin signaling at the level of PI3K, independently of IR.\\u003c/p\\u003e \\u003cp\\u003eWe then compared the relative potency of 5-HT and insulin for AKT phosphorylation \\u003cem\\u003ein vivo\\u003c/em\\u003e using LIRKO (Alb-Cre; \\u003cem\\u003eInsr\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e) mice. In WT mice, portal vein injection of either 5-HT or insulin induced hepatic AKT phosphorylation, but the response to 5-HT was weaker than that to insulin. Interestingly, 5-HT induced AKT phosphorylation was augmented in LIRKO livers compared with WT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eg, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ei), suggesting enhanced 5-HT/HTR2A signaling in the absence of insulin signaling. In contrast, 5-HT failed to induce AKT phosphorylation in \\u003cem\\u003eHtr2a;Insr\\u003c/em\\u003e double knockout (\\u003cem\\u003eHtr2a;Insr\\u003c/em\\u003e LKO) mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eg, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ei). Collectively, these results demonstrate that 5-HT activates hepatic PI3K-AKT-SREBP1 axis through HTR2A independently of IR.\\u003c/p\\u003e \\u003cp\\u003eHTR2A is a Gq-coupled receptor that activates phospholipase C, generating inositol 1,4,5-triphosphate (IP3) and diacylglycerol\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. IP3 triggers Ca\\u0026sup2;⁺ release from the endoplasmic reticulum, and Ca\\u0026sup2;⁺-bound calmodulin can activate PI3K\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e. Consistent with this model, blocking IP3 receptor signaling with 2-APB or chelating intracellular Ca\\u0026sup2;⁺ with BAPTA-AM reduced 5-HT induced AKT phosphorylation in AML-12 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eh,i). Inhibition of calmodulin with chlorpromazine (CPZ) also suppressed 5-HTinduced AKT phosphorylation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ej). These findings suggest that 5-HT/HTR2A signaling elevates intracellular Ca\\u0026sup2;⁺, enabling Ca\\u0026sup2;⁺/calmodulin dependent PI3K activation, AKT phosphorylation, and downstream SREBP1 activation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ek).\\u003c/p\\u003e \\u003cp\\u003eTo delineate the relative contributions of 5-HT and insulin signaling to hepatic lipogenesis under insulin resistant conditions, we examined the effects of 5-HT in the absence of HTR2A and/or IR in mice fed HFD. Hepatocyte specific gene deletion was achieved by AAV8-albumin-Cre in \\u003cem\\u003eHtr2a\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e (AAV-2A LKO), \\u003cem\\u003eInsr\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e (AAV-IR LKO), and \\u003cem\\u003eHtr2a\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e; \\u003cem\\u003eInsr\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e (AAV-DR LKO) mice. One week after AAV8 injection, the mice were fed HFD for 8 weeks to induce hepatic steatosis and systemic insulin resistance. Efficient gene deletion was confirmed by hepatic mRNA and protein analyses (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea,b).\\u003c/p\\u003e \\u003cp\\u003eIn this HFD induced insulin resistant state, both SREBP1 expression and AKT phosphorylation were reduced in AAV-2A LKO and AAV-DR LKO livers compared with WT controls, whereas no significant changes were observed in AAV-IR LKO livers (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea,b). Concordantly, histological analysis using H\\u0026amp;E and Oil Red O staining revealed marked reduction in lipid accumulation in AAV-2A LKO and AAV-DR LKO livers, accompanied by decrease in hepatic triglyceride content (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec). Body weight and plasma insulin levels were comparable among experimental groups (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed,e), excluding differences in systemic metabolic status as confounding factors.\\u003c/p\\u003e \\u003cp\\u003eTaken together, these results demonstrate that the enhanced hepatic lipogenesis and lipid accumulation under HFD induced insulin resistance are driven primarily by 5-HT/HTR2A signaling rather than insulin signaling.\\u003c/p\\u003e \\u003cp\\u003eImpaired suppression of hepatic gluconeogenesis is another defining feature of selective hepatic insulin resistance and has been attributed, at least in part, to reduced AKT mediated phosphorylation of FOXO1, resulting in sustained transcription of gluconeogenic genes\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. To assess the contribution of 5-HT to increased hepatic gluconeogenesis during insulin resistance, we examined FOXO1 phosphorylation in our models. Interestingly, FOXO1 phosphorylation was preserved in HFD fed WT mice and was instead reduced in HFD fed \\u003cem\\u003eHtr2a\\u003c/em\\u003e LKO mice (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea), indicating that 5-HT/HTR2A signaling contributes to the maintenance of AKT mediated FOXO1 phosphorylation under insulin resistant conditions. These findings argue against FOXO1 de-repression as the primary driver of gluconeogenesis in insulin resistance and suggest the existence of a FOXO1 independent mechanism that promotes hepatic gluconeogenesis.\\u003c/p\\u003e \\u003cp\\u003eCREB is a transcription factor that promotes gluconeogenic gene expression during fasting\\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e. Intriguingly, HFD increased CREB phosphorylation in both WT and \\u003cem\\u003eHtr2a\\u003c/em\\u003eLKO mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb), implicating CREB as a potential mediator for increased gluconeogenesis independent of FOXO1. Consistent with this notion, direct portal injection of 5-HT increased CREB phosphorylation in WT and \\u003cem\\u003eHtr2a\\u003c/em\\u003e LKO livers but not in \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO livers (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb,c, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec-f), demonstrating that 5-HT activates CREB via HTR2B.\\u003c/p\\u003e \\u003cp\\u003eWe next sought to identify signaling pathway linking HTR2B activation to CREB phosphorylation. Under physiological conditions, glucagon induces CREB phosphorylation through the adenylyl cyclase\\u0026ndash;cAMP\\u0026ndash;PKA pathway\\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e. However, pharmacological inhibition of PKA with KT-5720 or adenylyl cyclase with KH-7 did not attenuate 5-HT induced CREB phosphorylation in AML-12 cells suggesting that 5-HT induces CREB phosphorylation through a PKA independent mechanism (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed,e). Given that 5-HT/HTR2B signaling in adipose tissue activates PKG rather than PKA\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e, we examined nitric oxide (NO)-cGMP-PKG pathway in AML-12 cells. Inhibition of PKG (KT-5823), guanylyl cyclase (ODQ), or NO synthase (ADMA) reduced 5-HT induced CREB phosphorylation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ef-h). In addition, inhibition of intracellular Ca\\u0026sup2;⁺ signaling with 2-APB or BAPTA-AM reduced CREB phosphorylation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ei,j). These results indicate that 5-HT/HTR2B signaling increases intracellular Ca\\u0026sup2;⁺, activates NO synthase\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e, leading to NO production that stimulates guanylyl cyclase-cGMP-PKG pathway\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e to promote CREB phosphorylation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ek).\\u003c/p\\u003e \\u003cp\\u003eTo evaluate the physiological relevance of hepatic 5-HT/HTR2B signaling \\u003cem\\u003ein vivo\\u003c/em\\u003e, we examined \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO mice after 8weeks of HFD feeding. Body weight and hepatic triglyceride content were comparable between WT and \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO mice, although baseline glucose tolerance was slightly improved at 15 minutes in \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO mice (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea-c). In contrast, after 8 weeks of HFD feeding, glucose tolerance was markedly improved in \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO mice without a corresponding improvement in systemic insulin sensitivity (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea,b). In addition, pyruvate tolerance tests revealed reduced hepatic glucose production in \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed), suggesting that improved glucose tolerance is attributed to reduced gluconeogenesis. At molecular level, HFD increased hepatic CREB phosphorylation in WT mice, which was abolished in \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO mice, whereas FOXO1 phosphorylation remained unchanged (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed). Correspondingly, hepatic mRNA expression of gluconeogenic genes including \\u003cem\\u003eG6pc\\u003c/em\\u003e, \\u003cem\\u003ePck1\\u003c/em\\u003e, and \\u003cem\\u003eFbp1\\u003c/em\\u003e, was decreased in HFD fed \\u003cem\\u003eHtr2b\\u003c/em\\u003e LKO mice, whereas lipogenic genes (\\u003cem\\u003eSrebf1, Acly, Acaca, Fasn, Scd1\\u003c/em\\u003e) remained unchanged (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee). Together, these results demonstrate that 5-HT/HTR2B signaling promotes hepatic gluconeogenesis through PKG\\u0026ndash;CREB signaling axis, while hepatic lipogenesis under HFD induced insulin resistance remains selectively driven by 5-HT/HTR2A signaling.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we identify 5-HT as a key determinant of selective hepatic insulin resistance by demonstrating that it regulates hepatic lipogenesis and gluconeogenesis under insulin resistance, independently of insulin signaling. We show that hepatic HTR2A activation promoted lipogenesis via the AKT-mTORC1-S6K pathway, leading to SREBP1 activation and induction of lipogenic gene expression. In parallel, hepatic HTR2B activation promoted gluconeogenesis through the guanylyl cyclase-cGMP-PKG signaling pathway, resulting in CREB phosphorylation and induction of gluconeogenic gene expression. These findings establish 5-HT signaling as an insulin independent alternative pathway that regulates hepatic metabolic control in insulin resistant states, providing a mechanistic explanation for the long-standing metabolic paradox of preserved lipogenesis despite impaired insulin action in the liver.\\u003c/p\\u003e \\u003cp\\u003eInsulin is the principal regulator of hepatic lipid and glucose metabolism, transmitting signals through the IR-IRS1/IRS2-PI3K-AKT axis to multiple downstream branches, including FOXO1 and mTORC1\\u003csup\\u003e1,6,9,28\\u003c/sup\\u003e. Genetic dissection of these signaling components has demonstrated that failure of AKT to phosphorylate and inactivate FOXO1 leads to increased gluconeogenesis in insulin resistance\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e. Likewise, impaired insulin induced AKT phosphorylation reduces SREBP1 activation and lipogenesis. Accordingly, mice lacking these signaling components consistently display increased hepatic glucose production and reduced lipogenesis, a phenotype characteristic of \\u0026lsquo;total hepatic insulin resistance\\u0026rsquo;.\\u003c/p\\u003e \\u003cp\\u003eHowever, these findings are inconsistent with the metabolic features observed in patients with type 2 diabetes and in insulin resistant animal models induced by HFD feeding or genetic defects, such as \\u003cem\\u003eob/ob\\u003c/em\\u003e and \\u003cem\\u003edb/db\\u003c/em\\u003e mice\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e. In these models, hepatic gluconeogenesis and lipogenesis are concomitantly increased, a paradoxical phenomenon termed \\u0026lsquo;selective hepatic insulin resistance\\u0026rsquo;. Although this paradox has been attributed to differential impairment of insulin signaling\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e, this concept cannot fully explain the persistent lipogenesis despite diminished insulin responsiveness\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. Unlike models of total insulin resistance, models of type 2 diabetes show elevated basal AKT phosphorylation despite reduced responsiveness to exogenous insulin\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e, suggesting the presence of additional signaling inputs that sustain both lipogenesis and gluconeogenesis.\\u003c/p\\u003e \\u003cp\\u003ePreviously, we demonstrated that liver-specific deletion of \\u003cem\\u003eHtr2a\\u003c/em\\u003e reduced lipogenic gene expression and ameliorated hepatic steatosis in HFD fed mice, suggesting 5-HT/HTR2A as such an additional signal. Here, we extend these observations by defining the molecular mechanisms through which 5-HT independently regulates hepatic lipogenesis and gluconeogenesis. We present that 5-HT/HTR2A activated the PI3K-AKT-mTORC1-SREBP1 pathway through Ca\\u003csup\\u003e2+\\u003c/sup\\u003e-calmodulin dependent mechanism that occurred independently of the IR. In parallel, we identify a distinct role for 5-HT/HTR2B in regulating hepatic gluconeogenesis. Upon binding to HTR2B, 5-HT initiated a Ca\\u003csup\\u003e2+\\u003c/sup\\u003e-NO-cGMP-PKG signaling cascade that led to CREB phosphorylation. Deletion of \\u003cem\\u003eHtr2b\\u003c/em\\u003e in hepatocytes decreased gluconeogenic gene expression and hepatic glucose production under HFD feeding. Importantly, this pathway operated independently of insulin mediated FOXO1 regulation\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e or glucagon mediated PKA signaling\\u003csup\\u003e\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e. Our findings highlight an important role of CREB in driving hepatic gluconeogenesis in insulin resistant state and are consistent with previous studies, demonstrating that CREB activity contributes to fasting hyperglycemia in \\u003cem\\u003edb/db\\u003c/em\\u003e diabetic mice in which CREB inhibition attenuated hepatic glucose production and normalized plasma glucose levels\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e. Our data place CREB downstream of 5-HT/HTR2B signaling, providing a mechanistic link between elevated portal 5-HT and dysregulated hepatic glucose production.\\u003c/p\\u003e \\u003cp\\u003eCollectively, these findings identified 5-HT as a dual regulator of hepatic lipid and glucose metabolism under insulin resistant conditions, acting through HTR2A and HTR2B to promote lipogenesis and gluconeogenesis, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef). Given that HFD feeding increases 5-HT production in the gut and \\u003cem\\u003eHtr2a\\u003c/em\\u003e expression in the liver\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e, serotonergic signaling likely compensates for impaired insulin action and becomes a dominant driver of hepatic lipogenesis and gluconeogenesis in HFD fed mice. Therefore, we propose a new model to explain \\u0026lsquo;selective hepatic insulin resistance\\u0026rsquo;, in which non-insulin serotonergic signaling becomes pathologically dominant when insulin action is diminished. This suggests that HTR2A and HTR2B could be promising therapeutic targets for MASLD, type 2 diabetes, and related metabolic diseases.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\"\\u003e\\n \\u003ch2\\u003eAnimals\\u003c/h2\\u003e\\n \\u003cdiv\\u003e\\n \\u003cp\\u003eThe male mice were bred and maintained in specific pathogen-free barrier facilities. The facilities were kept under iso-temperature and iso-humidity conditions with a 12-hour light/dark cycle. The mice had \\u003cem\\u003ead libitum\\u003c/em\\u003e access to diet and water. The animal experiments were approved by the Institutional Animal Care and Use Committee at the Korea Advanced Institute of Science and Technology. All experiments were performed under guidelines and regulations. To induce diet induced insulin resistance model, 12, 13-week-old mice were adjusted to high-fat diet (HFD; 60% kcal fat, D12492, Research Diets) for 8 weeks. For normal chow diet (NCD) group, the mice were provided standard chow diet during same period to compare the effect of HFD induced insulin resistance. 12-week-old floxed mice (\\u003cem\\u003eHtr2a\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eInsr\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e and \\u003cem\\u003eHtr2a\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e;\\u003cem\\u003eInsr\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003ef/f\\u003c/em\\u003e\\u003c/sup\\u003e) were used to inject AAV which has Alb-Cre plasmid (CV17208-AV8, Charles River) at 3ⅹ10\\u003csup\\u003e12\\u003c/sup\\u003e GC/kg via tail vein to express hepatocyte specific Cre. After one week from injection, the mice were adjusted to HFD for 8 weeks. At the end of the experiments, mice were anesthetized \\u003cem\\u003ead libitum\\u003c/em\\u003e, after which blood samples were collected for subsequent analyses. Following blood collection, mice were euthanized and tissues were rapidly harvested. The collected tissues were either frozen and stored at -80℃ or immediately processed for downstream experimental procedures.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003eCell culture\\u003c/h3\\u003e\\n\\u003cdiv\\u003e\\n \\u003cp\\u003eAML-12 cell line (CRL-2254, ATCC) were cultured in Dulbecco\\u0026rsquo;s Modified Eagle Medium (DMEM, SH30243.01, Hyclone) supplemented with 100 \\u0026micro;g/ml penicillin/streptomycin (15140-122, Gibco) and 10% Fetal Bovine Serum (16000-044, Gibco) at 37 ℃ in a humidified 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e atmosphere. Cell culture media was changed every 48 hours and subcultured at 70\\u0026ndash;80% confluence using 0.25% trypsin-EDTA solution (25200-056, Gibco). Cells at 80\\u0026ndash;90% confluence were used in experiments. Cells were treated with 100nM 5-HT (H9523, sigma), MK2206 (HY-10358, MedCamExpress), Wortmannin (681675, sigma), S961 (051\\u0026ndash;86, Phoenix pharm), 2-APB (100065, sigma), BAPTA-AM (A1076, sigma), Chlorpromazine (CPZ, 31679, Supelco), KH-7 (K3394, sigma), KT-5720 (420320, sigma), ODQ (495320, sigma), KT-5823 (420321, sigma) or ADMA (D4268, sigma). Cells were pretreated with the indicated inhibitors for 30 minutes before 5-HT treatment. Before experiments, cells for AKT and SREBP1 induction analysis were serum deprived for 6 hours, while those for CREB activation were serum deprived for 2 hours.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003ePortal vein injection\\u003c/h3\\u003e\\n\\u003cdiv\\u003e\\n \\u003cp\\u003eMice for portal vein injection were anesthetized via intraperitoneal injection of avertin with combination of isoflurane inhalation. The peritoneum was opened by a midline incision and the portal vein was exposed. Insulin syringe (BD, 328820) with 100 \\u0026micro;l PBS, 20 ng 5-HT in 100 \\u0026micro;l PBS or 2 ng insulin in 100 \\u0026micro;l PBS was carefully inserted into the portal vein. After 5 minutes, liver tissue was harvested and mice were sacrificed. Before injection, the mice designated for AKT phosphorylation analysis were fasted for 16 hours, while those for CREB phosphorylation were fasted for 4 hours.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003eWesternblot analysis\\u003c/h3\\u003e\\n\\u003cdiv\\u003e\\n \\u003cp\\u003eLiver tissue and fully cultured AML-12 cells were extracted by RIPA-Lysis extraction buffer (89901, Thermo scientific) containing protease inhibitor (P3100-010, GenDEPOT) and phosphatase inhibitor (P3200-010, GenDEPOT). Tissue samples in bead tubes were homogenized by Fast Prep-24 (MP Biomedicals).\\u003c/p\\u003e\\n \\u003cp\\u003eAfter protein extraction, suspensions were centrifuged at 13000 rpm at 4 ℃ for 15 minutes. To determine protein concentration, the BCA Protein Assay Kit (23227, Pierce) was performed; 10\\u0026ndash;20 \\u0026micro;g of protein was subjected to 8\\u0026ndash;10% SDS-PAGE gels and transferred to the PVDF membranes (IPVH00010, Millipore) which are activated by methanol. Transferred membranes were incubated in the blocking solution (TBS buffer with 0.1% Tween 20) containing 5% BSA (w/v ratio) (0210370380, Mpbio) for 1 hour at room temperature. All antibodies used in this study are listed in Supplementary Table. Primary antibodies were reacted overnight at 4 ℃; secondary antibodies were reacted for 1 hour 30 minutes at room temperature. ECL substrate (WBKLS0500, Millipore) was used to amplify the detectable signals for ImageQuant 800 (cytiva, Amercham). The band intensities were quantified and represented in bar graphs.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003eQuantitative PCR (qPCR) analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eFor mRNA extraction to examine transcriptional levels, liver tissue samples in bead tube or treated cells were collected in Tri-RNA reagent (15596-018, Ambion) and homogenized by Fast Prep-24 (MP Biomedicals). And extraction protocol, which includes chloroform (67-66-3, Junsei) and iso-propyl alcohol (67-63-0, Supelco), was used. After extraction, cDNA was synthesized from 1 \\u0026micro;g of total RNA using a High Capacity of cDNA Reverse Transcriptase Kit (4368813, Applied Biosystems, Foster Cuy, CA, USA).\\u003c/p\\u003e\\n\\u003cdiv\\u003e\\n \\u003cp\\u003eQuantitative real time-polymerase chain reaction (qPCR) was performed on a ViiA7 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using Fast SYBR Green Master Mix (4385612, Applied Biosystems) according to the manufacturer\\u0026rsquo;s instructions. Expressional profiles were quantified according to the relative ddCt method using 36B4 as a reference gene. The sequence of primers used in this study are listed in Supplementary Table.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec8\\\"\\u003e\\n \\u003ch2\\u003eHematoxylin \\u0026amp; Eosin (H\\u0026amp;E) staining\\u003c/h2\\u003e\\n \\u003cp\\u003eFor H\\u0026amp;E staining, harvested liver tissues were fixed in formalin and embedded in paraffin. Five-micron-thick sections were deparaffinized, rehydrated, and stained with H\\u0026amp;E (AR173, Agilent Dako, Santa Clara, CA, USA), according to the manufacturer\\u0026rsquo;s instruction.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003eOil red O staining\\u003c/h3\\u003e\\n\\u003cp\\u003eFor Oil red O staining, harvested liver tissues were fixed in 4% paraformaldehyde for 4 hours and dehydrated serially by 20% sucrose and 40% sucrose solution. Dehydrated tissues are frozen in Tissue Tek. O.C.T compound (4583, Sakura).\\u003c/p\\u003e\\n\\u003cp\\u003eThe frozen liver sections were cut at 8\\u0026ndash;10 micron-thick and dry at tabletop. After washing by tap water for 5 minutes, the sections were rinsed with 60% isopropanol. Prepared Oil red O working solution, which is mixture of 0.5% Oil red O stock solution in isopropanol with distilled water (DW) at 6:4 ratio, was used to stain the rinsed section for 15 minutes. After staining, the section was rinsed with 60% isopropanol and stained with hematoxylin for 10 seconds to stain nuclei. Then, rinsed sections by DW were mounted with a medium under the cover glass. Images were acquired by bright-field microscope (Nikon eclipse Ni).\\u003c/p\\u003e\\n\\u003ch3\\u003eGlucose/Pyruvate/Insulin tolerance test (GTT/PTT/ITT)\\u003c/h3\\u003e\\n\\u003cdiv\\u003e\\n \\u003cp\\u003eFor glucose tolerance test (GTT), the mice were fasted overnight for 16 hours and 2 g/kg D-glucose (20%, Dai han pharm) was injected in mice intraperitoneally. For glucose measurement, blood glucose levels from 0, 15, 30, 60, 90 and 120 minutes after injection were measured from tail vein using a glucometer (GlucoDr.TOP glucometer, Allmedicus).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003eFor pyruvate tolerance test (PTT) and insulin tolerance test (ITT), the mice were fasted for 6 hours and 2 g/kg-sodium pyruvate (P5280, Sigma) and 1 U/kg insulin (I9278, Sigma), respectively, was injected in mice intraperitoneally. For glucose measurement, blood glucose levels were measured from tail vein using a glucometer (GlucoDr.TOP glucometer, Allmedicus) at indicated time points after injection.\\u003c/p\\u003e\\n\\u003cdiv id=\\\"Sec11\\\"\\u003e\\n \\u003ch2\\u003eQuantification of hepatic triglycerides (TG) level\\u003c/h2\\u003e\\n \\u003cdiv\\u003e\\n \\u003cp\\u003eLiver tissues were homogenized in 5% NP-40 using Fast Prep-24 (MP Biomedicals). TGs were solubilized by two cycles of heating homogenates to 95\\u0026deg;C for 5 minutes and then, cooling on ice. Triglyceride Reagent (T2449, sigma) or DW was added, and TGs were hydrolyzed into glycerol by incubating samples at 37\\u0026deg;C for 30 minutes. For colorimetric assay of hydrolyzed TG levels, samples were incubated with Free Glycerol Reagent (F6428, sigma) at 37\\u0026deg;C for 5 minutes. Differences in absorbance at 540 nm between hydrolyzed and non-hydrolyzed TGs were quantified using a glycerol standard (G7793, sigma). TG content was normalized to the protein concentration in homogenates, measured using a BCA Protein Assay Kit (23227, Pierce).\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec12\\\"\\u003e\\n \\u003ch2\\u003eInsulin ELISA\\u003c/h2\\u003e\\n \\u003cdiv\\u003e\\n \\u003cp\\u003ePlasma insulin concentrations were measured using insulin immunoassay kit (#80-INSMSU, ALPCO) according to the manufacturer\\u0026rsquo;s instructions. Briefly, whole blood was collected into EDTA-coated tubes, and centrifuged at 2,000 g for 5 minutes at 4\\u0026deg;C to separate plasma. Plasma samples were aliquoted and stored at -80\\u0026deg;C until analysis. Prior to assay samples were thawed on ice and briefly centrifuged to remove precipitates. All standards, controls and samples were assayed in duplicate.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec13\\\"\\u003e\\n \\u003ch2\\u003eQuantification and statistical analysis\\u003c/h2\\u003e\\n \\u003cdiv\\u003e\\n \\u003cp\\u003eValues from every experiment are expressed as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard error of mean (SEM). To compare groups, the two-tailed Student\\u0026rsquo;s t test or one-way analysis of variance (ANOVA) followed by post-hoc Tukey\\u0026rsquo;s test were used. P values below 0.05 were considered statistically significant. The levels of significance indicated in the graphs are *; \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, **; \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01, ***; \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001, ****; \\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT, Ministry of Science and ICT) (No. RS-2025-00513814 and No. RS-2025-02214748), the Korea Health Technology R\\u0026amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health \\u0026amp; Welfare, Republic of Korea (No. RS-2025-02262990) (to H.K.), Inha University (No. 71580 to Y.A.M.) and the National Research Foundation of Korea (NRF) (No. RS-2022-00166199 to I.H., No. RS-2023-00247558 to W.G.C, No. RS-2024-00408919 to W.C., and No. RS-2024-00453271 to W.I.C.).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eI.H., J.E.N., Y.M. and H.K. conceived the study. I.H., W.G.C., W.C., W.I.C. and J.E.N. performed experiments. J.E.N., Y.M. and H.K. wrote the manuscript. H.K. and Y.M supervised the project. I.H. and W.C. edited the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eBo, T.\\u003cem\\u003e\\u0026nbsp;et al.\\u003c/em\\u003e Hepatic selective insulin resistance at the intersection of insulin signaling and metabolic dysfunction-associated steatotic liver disease. \\u003cem\\u003eCell Metab.\\u003c/em\\u003e 36, 947-968 (2024). https://doi.org:10.1016/j.cmet.2024.04.006\\u003c/li\\u003e\\n \\u003cli\\u003eStefan, N., Yki-Jarvinen, H. \\u0026amp; Neuschwander-Tetri, B. A. 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Nutrient control of phosphorylation and translocation of FoxO1 in C57BL/6 and db/db mice. \\u003cem\\u003eInt. J. Mol. Med.\\u003c/em\\u003e 18, 433-439 (2006).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-portfolio\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Nature Portfolio\",\"twitterHandle\":\"\",\"acdcEnabled\":false,\"dfaEnabled\":false,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8819291/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8819291/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eSelective hepatic insulin resistance is characterized by preserved lipogenesis despite the failure to suppress gluconeogenesis, yet the detailed molecular mechanisms underlying this paradox remain incompletely understood. Here, we identify serotonin as an insulin independent regulator that differentially regulates hepatic lipid and glucose metabolism in insulin resistant states. Serotonin promoted hepatic lipogenesis through HTR2Amediated activation of the Ca\\u003csup\\u003e2+\\u003c/sup\\u003e-PI3K-AKT-mTORC1 signaling cascade, leading to SREBP1 activation independently of insulin receptor. In parallel, serotonin signaling through HTR2Bstimulates hepatic gluconeogenesis via a Ca\\u003csup\\u003e2+\\u003c/sup\\u003e-NO-cGMP-PKG pathway, resulting in CREB phosphorylation independently of PKA. Genetic disruption of HTR2A selectively attenuated hepatic lipogenesis, whereas deletion of HTR2B suppressed gluconeogenic gene expression and hepatic glucose production without affecting lipogenesis. Together, these findings establish serotonergic regulation as a dual, insulin independent driver of hepatic lipogenesis and gluconeogenesis under insulin resistant conditions, providing a mechanistic explanation for selective hepatic insulin resistance and identifying serotonin receptors as potential therapeutic targets for metabolic disease.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Serotonin drives selective hepatic insulin resistance via HTR2A and HTR2B\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-03-04 05:40:28\",\"doi\":\"10.21203/rs.3.rs-8819291/v1\",\"editorialEvents\":[],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-communications\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"NCOMMS\",\"sideBox\":\"Learn more about [Nature Communications](http://www.nature.com/ncomms/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://mts-ncomms.nature.com/\",\"title\":\"Nature Communications\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature Communications\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"a7cb2338-b90c-41b9-9d2e-6c05347ebea7\",\"owner\":[],\"postedDate\":\"March 4th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":62942096,\"name\":\"Biological sciences/Cell biology/Cell signalling/Hormone receptors\"},{\"id\":62942097,\"name\":\"Biological sciences/Cell biology/Cell signalling/Insulin signalling\"},{\"id\":62942098,\"name\":\"Biological sciences/Cell biology/Mechanisms of disease\"}],\"tags\":[],\"updatedAt\":\"2026-03-04T05:40:28+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-03-04 05:40:28\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8819291\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8819291\",\"identity\":\"rs-8819291\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}