TLR4 Deficiency Fails to Protect Against Diet-Induced Behavioral Impairments Under High-Sucrose Feeding

TLR4 Deficiency Fails to Protect Against Diet-Induced Behavioral Impairments Under High-Sucrose Feeding | 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 Short Report TLR4 Deficiency Fails to Protect Against Diet-Induced Behavioral Impairments Under High-Sucrose Feeding Ghadeer Alhamar, Rawan Najem, Texy Jacob, Steve Shenouda, Fawaz Alzaid, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7620609/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Obesity and type 2 diabetes (T2D) are accompanied by systemic and neural inflammation that contribute to cognitive and behavioral impairments. Toll-like receptor 4 (TLR4) links nutrient excess to inflammatory signaling and has been implicated in both metabolic dysfunction and neurocognitive decline. While TLR4 deficiency offers protection against high-fat diet (HFD) induced obesity and insulin resistance, the role of dietary sucrose in shaping TLR4 mediated behavioral outcomes remains unclear. We compared wild-type (WT) and TLR4 knockout (KO) mice fed chow, a normal sucrose HFD (NS-HFD), or a high sucrose HFD (HS-HFD) for 13 weeks. TLR4 KO mice on NS-HFD exhibited reduced weight gain, improved insulin sensitivity, and enhanced exploratory behavior with reduced anxiety-like activity in an open field test. However, these protective metabolic and behavioral effects were abolished under HS-HFD, where TLR4 KO and WT mice performed similarly. These findings reveal that high dietary sucrose overrides TLR4 dependent protection, underscoring critical gene and diet interactions in obesity and cognitive dysfunction. TLR4KO Anxiety-like behavior Obesity Insulin Resistance High fat diet with high sucrose Figures Figure 1 Figure 2 Significance Statement This study demonstrates that high dietary sucrose abolishes the metabolic and behavioral protection normally conferred by TLR4 deficiency, revealing a critical diet–gene interaction that links obesity to cognitive dysfunction. 1.0 Introduction Obesity and type 2 diabetes (T2D) are characterized not only by metabolic disorder, but also by chronic low-grade inflammation, contributing to systemic and neural dysfunction ( 1 , 2 ). Toll-like receptor 4 (TLR4), a pattern recognition receptor of the innate immune system, plays a central role in this process ( 3 ). TLR4 is activated by dietary lipids, such as saturated fatty acids, and bacterial lipopolysaccharides (LPS), triggering downstream MyD88- and TRIF-dependent signaling cascades that lead to the production of pro-inflammatory cytokines ( 4 – 6 ). Through these mechanisms, TLR4 acts as a molecular link between overnutrition, inflammation, and insulin resistance. High fat diets (HFD) are widely used in rodent models to induce insulin resistance, hepatic steatosis and obesity ( 7 ). Studies in TLR4 knockout (TLR4 KO) mice have shown partial protection against diet-induced metabolic dysfunction and weight gain, highlighting the importance of TLR4 in the progression of these metabolic disorders ( 8 , 9 ). Beyond metabolic dysfunction, diet-induced obesity and insulin resistance have been increasingly associated with alterations in the central nervous system and brain function, resulting in neurocognitive dysfunction, including anxiety, depression and memory discrepancies ( 10 ). While much attention has focused on the role of HFDs in TLR4-mediated obesity, inflammation, and metabolic dysfunction, far less is known about the impact of diets combining high fat with high sucrose (HS). Emerging evidence suggests that sucrose itself can drive inflammatory changes ( 11 ). Both overnutrition and sucrose-rich diets are implicated in early cognitive deficits. Rodent models have demonstrated changes in locomotor activity, energy balance and anxiety-like behaviors ( 12 , 13 ). In fact, one study on the impact of HFD on TLR4 KO mice showed that TLR4 KO mice performed similarly in a Morris water maze and found the platform in both the HFD and normal diet group, where wild-type mice on the HFD took significantly longer, indicating a decline in cognitive function ( 14 ). Although extensive evidence links TLR4 and HFDs to metabolic dysfunction and behavioral changes, the contribution of HS remains unclear. This study attempts to address this gap by investigating the impacts of HS-HFDs on TLR4 -mediated cognitive dysfunction. 2.0 Methods 2.1 Animals C57BL/6 and TLR4 KO mice raised in the Animal Core Facility of Dasman Diabetes Institute were purchased from Jackson Laboratory. Mice were maintained ad libitum on a standard chow diet and housed in a temperature-controlled facility (23°C) under a 12-h light/12-h dark cycle. Experiments were conducted in mice aged 7–9 weeks, which were randomly assigned into three groups (N = 5 per group). The experimental groups included: 1) standard chow diet, 2) palmitate high-fat diet (D18060404i, Research Diets Inc.) supplemented with 6.5% sucrose in the drinking water (normal sucrose)(NS-HFD), and 3) palmitate high-fat diet with high sucrose content (D21042005i, Research Diets Inc.)(HS-HFD). Body weight and food intake were monitored weekly throughout the study. At 7–8 weeks of feeding mice were placed in metabolic cages, and at 10 weeks of feeding an open field test (OFT) was performed. Moreover, at weeks 12 and 13 of feeding, insulin tolerance tests (ITT) and oral glucose tolerance tests (OGTT) were performed, respectively. After 14 weeks, mice were sacrificed, and tissues and organs were collected. 2.2 Open field test Open field test (OFT) was used to assess anxiety-like behavior and locomotion using an open field box (WidthxLengthxDepth = 50cmx50cmx50cm) and a digital camera housed about the open field box. The camera was connected to a computer and had a video-tracking system (ANY-Maze, Stoelting Co, IL-USA) which records and measures all movements. Mice were placed in the behavioral test room for 1 hour before the test to allow them to acclimate before being placed in the chamber. Mice left to freely roam in the chamber for 30 minutes, with their movements being recorded. The distance travelled, number of entries and time spent in the peripheral and central zone were assessed. 2.3 Insulin and glucose tolerance tests To perform an ITT, the mice were fasted for 4 hours, once baseline (0 minute) glucose measurements were taken from all mice, insulin was administered intraperitoneally at a dose of 0.5U/kg. Blood glucose levels were then measured at 15, 30, 45, 60, 75, 90, 105, and 120 minutes ( 15 , 16 ). For the OGTT, mice were fasted for 12 hours. After baseline blood glucose levels (0 minutes), glucose was administered orally to each mouse at a dose of 1g/kg, followed by glucose levels being measured at 15, 30, 45, 60, 75, 105, and 120 minutes ( 15 , 16 ). 2.4 Statistical analysis Data was presented as mean ± SEM, unless otherwise indicated, outliers were detected and removed based on a ROUT test. D’Agostino-Pearson omnibus normality test was performed to determine normal distribution, for normally distributed sample sets a Student’s T-test was performed or Two-way ANOVA, for non-normally distributed sample sets a Mann-Whitney test was performed to determine p-values. P-values < 0.05 were considered significant, ns represents no significance. All statistical analyses were performed using GraphPad prism (La Jolla, CA, USA, version 10.2.0). 3.0 Results In order to assess the role of TLR4 deficiency under different dietary conditions, WT and TLR4 KO mice (aged 8–9 weeks) were placed on either chow, NS-HFD or HS-HFD for 13 weeks (Fig. 1 ). By the end of the feeding period, body weight analysis showed that TLR4 KO mice gained significantly less weight when fed NS-HFD compared to the WT, while no significant differences were seen in either chow or HS-HFD groups (Fig. 1 C-F). Glucose homeostasis was then assessed by ITT and OGTT. Consistent to the reduced weight gain, TLR4 KO mice on NS-HFD presented with improved insulin sensitivity and glucose tolerance compared to WT mice, whereas no differences were observed in the HS-HFD group (Fig. 1 G-H). We next assessed whether TLR4 KO influenced exploratory behavior under different dietary conditions using an OFT (Fig. 2 ). Representative locomotor traces illustrate the differences in exploratory activity between WT and TLR4 KO mice under chow, NS-HFD or HS-HFD (Fig. 2 A). Under chow and HS-HFD, WT and TLR4 KO mice show comparable movement patterns, however under NS-HFD TLR4 KO mice show visibly greater locomotor activity, including frequent entries into the central zone. Additionally, TLR4 KO mice showed a significant increased in total distance travelled, accompanied with a higher percentage of time in the central zone and overall more frequent central zone entries when fed NS-HFD compared to the WT. However, this significance was lost in TLR4 KO fed HS-HFD (Fig. 2 B-D). These behavioral differences may suggest that TLR4 KO mice fed NS-HFD present with reduced anxiety-like behavior and enhanced exploratory drive. In contrast, when mice were fed HS-HFD, bot the WT and TLR4 KO mice has comparable locomotor patterns, indicating that the behavioral phenotype associated with TLR4 deficiency is diet specific rather than a generalized effect. 4.0 Discussion Consistent with previous studies, we found that TLR4 KO protects against diet induced adiposity and insulin resistance under NS-HFD or standard HFD ( 8 , 9 ). TLR4 KO mice exhibited significantly less weight gain, and improved glucose handling compared to WT controls, this is in line with the established role of TLR4 in mediating lipid-induced inflammation and metabolic dysfunction. These findings reinforce the concept that TLR4 is a key sensor that links dietary fats to systemic metabolic outcomes. Beyond metabolism, previous studies have assessed the role of TLR4 in cognitive brain function and behavior. Fei et al. demonstrated that TLR4 -/- mice exhibit improved memory abilities and learning, with attenuated fear response ( 17 ). Similarly, neuronal TLR4 deletion in male mice confers protection against ethanol induced anxiety behaviors ( 18 ). Additionally, HFD and high cholesterol fed WT C57BL/6J mice were associated with a depressive and anxiety like state mediated by an elevated expression of TLR4 ( 19 ). Together, these studies point a role for TLR4 in shaping neurobehavioral responses to dietary and environmental stressors. A novel aspect of our study is the demonstration that this protective effect of TLR4 deficiency is abolished when high sucrose is added into the HFD (HS-HFD). Mice fed HS-HFD gained weight at levels comparable to WT mice and showed no improvement in insulin or glucose sensitivity. This suggests that the detrimental effects of excess sucrose can override the beneficial impacts of TLR4 KO. Importantly, we also observed changes in behavior in TLR4 KO mice fed a NS-HFD or a HS-HFD. Where TLR4 KO mice fed NS-HFD displayed enhanced exploratory activity and reduced anxiety compared to the WT, this phenotype was abolished under HS-HFD. In fact, both mice strains demonstrated restricted central zone exploration, suggesting that not only can the addition of high sucrose to a HFD diminish metabolic protection, but may also promote anxiety-like behaviors in TLR4 KO mice. Overall, these findings extend prior work by showing that the interaction between dietary fat and sugar fundamentally alters the contribution of innate immune signaling to both metabolic and behavioral outcomes. This highlights the importance of diet composition, and not simply fat alone, that can shape physiology, suggesting that increased sucrose may instigate neurobiological and inflammatory pathways that bypass TLR4. Future studies will be needed to define these exact mechanisms, but our results suggest a close interplay between nutrition, immune signaling, and neurobehavioral health. Abbreviations T2D, Type 2 Diabetes TLR4, Toll-like receptor 4 TLR4 KO, Toll-like receptor 4 knockout LPS, Lipopolysaccharides NS-HFD, normal sucrose high fat diet HS-HFD, high sucrose high fat diet ITT, insulin tolerance test OGTT, oral glucose tolerance test OFT, open field test Declarations Ethics approval Ethical approval for this study was obtained from the Animal Care and Use Committee based in Dasman Diabetes Institute Competing interests The authors declare no conflict of interest. Funding This work was supported by funding from the Kuwait Foundation for the Advancement of Science (KFAS), grant no. (RA AM-2020-007; RA-AM 2023-021). Authors' contributions GA and RA generated the study, GA curated data and analysis and wrote the first draft. RN performed and assisted in behavioral tests, TJ and SS managed mice cohorts and assisted in data collection, FZ and FA-M reviewed the manuscript, edited and analyzed data. Data Availability All data generated or analysed during this study are included in this article. Acknowledgements NA References Mukherjee S, Kundu R, Vidaković M. Editorial: Metaflammation in obesity and diabetes. Front Endocrinol (Lausanne). 2024;15:1540999. Niedowicz DM, Reeves VL, Platt TL, Kohler K, Beckett TL, Powell DK, et al. Obesity and diabetes cause cognitive dysfunction in the absence of accelerated β-amyloid deposition in a novel murine model of mixed or vascular dementia. Acta Neuropathol Commun. 2014;2:64. Kumar S, Sharma V, Yadav S. TLR4 Targeting: A Promising Therapeutic Approach Across Multiple Human Diseases. Curr Protein Pept Sci. 2025;26(4):241–58. 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TLR4 Deletion Improves Cognitive Brain Function and Structure in Aged Mice. Neuroscience. 2022;492:1–17. Li Y, Zhu S, Xie K, Feng X, Chen L, Wu X, et al. TLR4 in Tph2 neurons modulates anxiety-related behaviors in a sex-dependent manner. Neuropharmacology. 2022;216:109175. Strekalova T, Evans M, Costa-Nunes J, Bachurin S, Yeritsyan N, Couch Y, et al. Tlr4 upregulation in the brain accompanies depression- and anxiety-like behaviors induced by a high-cholesterol diet. Brain Behav Immun. 2015;48:42–7. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted 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. 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1","display":"","copyAsset":false,"role":"figure","size":647033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiet information and demographic characteristics of mice cohorts. A) \u003c/strong\u003eExperimental timeline: WT and TLR4 KO mice (8-9 weeks) were placed in either chow, NS-HFD or HS-HFD (N=5). At 19 weeks (10 weeks of feeding), behavioral tests were performed, specifically open field test, followed by an ITT at week 20, and OGTT at week 21. \u003cstrong\u003eB) \u003c/strong\u003eShows the macronutrient content of both the NS-HFD and HS-HFD. \u003cstrong\u003eC)\u003c/strong\u003e Final body weight of WT and TLR4 KO mice after 13 weeks being fed chow, NS-HFD or HS-HFD. \u003cstrong\u003eD-F)\u003c/strong\u003e Percent body weight gain over time between WT and TLR4 KO on chow (D), NS-HFD (E), and HS-HFD (F). TLR4 KO mice gained significantly less weight than WT on NS-HFD, but not under chow or HS-HFD. \u003cstrong\u003eG) \u003c/strong\u003eInsulin tolerance test (ITT) curves for WT and TLR4 KO mice under each diet condition. \u003cstrong\u003eH) \u003c/strong\u003eOral glucose tolerance test (OGTT) curves for WT and TLR4 KO mice under each diet condition. Data are shown as mean ± SEM. P-values were calculated via Two-way ANOVA, Student’s T-test or Mann-Whitney test where appropriate. *P-value \u0026lt;0.05, **\u0026lt; 0.01, ***\u0026lt;0.001, ns= non-significant.\u003c/p\u003e","description":"","filename":"floatimage121.png","url":"https://assets-eu.researchsquare.com/files/rs-7620609/v1/2b7965b683e03fbd2ac992b1.png"},{"id":94457064,"identity":"9723a01f-62bc-47b1-9591-3e5dd2fa8817","added_by":"auto","created_at":"2025-10-27 14:45:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1222612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOpen field test reveals diet-specific effects of TLR4 deficiency on exploratory behavior. A) \u003c/strong\u003eRepresentative locomotor activity traces of WT and TLR4 KO mice under chow, NS-HFD, and HS-HFD conditions. \u003cstrong\u003eB) \u003c/strong\u003eShows the total distance travelled, \u003cstrong\u003eC) \u003c/strong\u003epercentage time spent in the central zone, and \u003cstrong\u003eD)\u003c/strong\u003e number of central zone entries. TLR4 KO mice on NS-HFD exhibited significantly greater locomotor activity and central exploration compared to WT mice, while no significant differences were observed under chow or HS-HFD conditions. Data are shown as mean ± SEM. *P-value \u0026lt;0.05, **\u0026lt; 0.01, ***\u0026lt;0.001, ns= non-significant.\u003c/p\u003e","description":"","filename":"floatimage215.png","url":"https://assets-eu.researchsquare.com/files/rs-7620609/v1/d3f9fbed063339a71319e208.png"},{"id":103852850,"identity":"a7793844-12fc-43b2-9c37-6910471f1c79","added_by":"auto","created_at":"2026-03-03 17:10:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2163505,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7620609/v1/53c49d95-caee-4024-b3b3-e176195c0275.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"TLR4 Deficiency Fails to Protect Against Diet-Induced Behavioral Impairments Under High-Sucrose Feeding","fulltext":[{"header":" Significance Statement","content":"\u003cp\u003eThis study demonstrates that high dietary sucrose abolishes the metabolic and behavioral protection normally conferred by TLR4 deficiency, revealing a critical diet–gene interaction that links obesity to cognitive dysfunction. \u003c/p\u003e"},{"header":"1.0 Introduction","content":"\u003cp\u003eObesity and type 2 diabetes (T2D) are characterized not only by metabolic disorder, but also by chronic low-grade inflammation, contributing to systemic and neural dysfunction (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Toll-like receptor 4 (TLR4), a pattern recognition receptor of the innate immune system, plays a central role in this process (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). TLR4 is activated by dietary lipids, such as saturated fatty acids, and bacterial lipopolysaccharides (LPS), triggering downstream MyD88- and TRIF-dependent signaling cascades that lead to the production of pro-inflammatory cytokines (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Through these mechanisms, TLR4 acts as a molecular link between overnutrition, inflammation, and insulin resistance.\u003c/p\u003e\u003cp\u003eHigh fat diets (HFD) are widely used in rodent models to induce insulin resistance, hepatic steatosis and obesity (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Studies in TLR4 knockout (TLR4 KO) mice have shown partial protection against diet-induced metabolic dysfunction and weight gain, highlighting the importance of TLR4 in the progression of these metabolic disorders (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Beyond metabolic dysfunction, diet-induced obesity and insulin resistance have been increasingly associated with alterations in the central nervous system and brain function, resulting in neurocognitive dysfunction, including anxiety, depression and memory discrepancies (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile much attention has focused on the role of HFDs in TLR4-mediated obesity, inflammation, and metabolic dysfunction, far less is known about the impact of diets combining high fat with high sucrose (HS). Emerging evidence suggests that sucrose itself can drive inflammatory changes (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Both overnutrition and sucrose-rich diets are implicated in early cognitive deficits. Rodent models have demonstrated changes in locomotor activity, energy balance and anxiety-like behaviors (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In fact, one study on the impact of HFD on TLR4 KO mice showed that TLR4 KO mice performed similarly in a Morris water maze and found the platform in both the HFD and normal diet group, where wild-type mice on the HFD took significantly longer, indicating a decline in cognitive function (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Although extensive evidence links TLR4 and HFDs to metabolic dysfunction and behavioral changes, the contribution of HS remains unclear. This study attempts to address this gap by investigating the impacts of HS-HFDs on TLR4 -mediated cognitive dysfunction.\u003c/p\u003e"},{"header":"2.0 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animals\u003c/h2\u003e\u003cp\u003eC57BL/6 and TLR4 KO mice raised in the Animal Core Facility of Dasman Diabetes Institute were purchased from Jackson Laboratory. Mice were maintained \u003cem\u003ead libitum\u003c/em\u003e on a standard chow diet and housed in a temperature-controlled facility (23\u0026deg;C) under a 12-h light/12-h dark cycle. Experiments were conducted in mice aged 7\u0026ndash;9 weeks, which were randomly assigned into three groups (N\u0026thinsp;=\u0026thinsp;5 per group). The experimental groups included: 1) standard chow diet, 2) palmitate high-fat diet (D18060404i, Research Diets Inc.) supplemented with 6.5% sucrose in the drinking water (normal sucrose)(NS-HFD), and 3) palmitate high-fat diet with high sucrose content (D21042005i, Research Diets Inc.)(HS-HFD). Body weight and food intake were monitored weekly throughout the study. At 7\u0026ndash;8 weeks of feeding mice were placed in metabolic cages, and at 10 weeks of feeding an open field test (OFT) was performed. Moreover, at weeks 12 and 13 of feeding, insulin tolerance tests (ITT) and oral glucose tolerance tests (OGTT) were performed, respectively. After 14 weeks, mice were sacrificed, and tissues and organs were collected.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Open field test\u003c/h2\u003e\u003cp\u003eOpen field test (OFT) was used to assess anxiety-like behavior and locomotion using an open field box (WidthxLengthxDepth\u0026thinsp;=\u0026thinsp;50cmx50cmx50cm) and a digital camera housed about the open field box. The camera was connected to a computer and had a video-tracking system (ANY-Maze, Stoelting Co, IL-USA) which records and measures all movements. Mice were placed in the behavioral test room for 1 hour before the test to allow them to acclimate before being placed in the chamber. Mice left to freely roam in the chamber for 30 minutes, with their movements being recorded. The distance travelled, number of entries and time spent in the peripheral and central zone were assessed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Insulin and glucose tolerance tests\u003c/h2\u003e\u003cp\u003eTo perform an ITT, the mice were fasted for 4 hours, once baseline (0 minute) glucose measurements were taken from all mice, insulin was administered intraperitoneally at a dose of 0.5U/kg. Blood glucose levels were then measured at 15, 30, 45, 60, 75, 90, 105, and 120 minutes (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). For the OGTT, mice were fasted for 12 hours. After baseline blood glucose levels (0 minutes), glucose was administered orally to each mouse at a dose of 1g/kg, followed by glucose levels being measured at 15, 30, 45, 60, 75, 105, and 120 minutes (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Statistical analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eData was presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, unless otherwise indicated, outliers were detected and removed based on a ROUT test. D\u0026rsquo;Agostino-Pearson omnibus normality test was performed to determine normal distribution, for normally distributed sample sets a Student\u0026rsquo;s T-test was performed or Two-way ANOVA, for non-normally distributed sample sets a Mann-Whitney test was performed to determine p-values. P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant, ns represents no significance. All statistical analyses were performed using GraphPad prism (La Jolla, CA, USA, version 10.2.0).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3.0 Results","content":"\u003cp\u003eIn order to assess the role of TLR4 deficiency under different dietary conditions, WT and TLR4 KO mice (aged 8\u0026ndash;9 weeks) were placed on either chow, NS-HFD or HS-HFD for 13 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By the end of the feeding period, body weight analysis showed that TLR4 KO mice gained significantly less weight when fed NS-HFD compared to the WT, while no significant differences were seen in either chow or HS-HFD groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-F). Glucose homeostasis was then assessed by ITT and OGTT. Consistent to the reduced weight gain, TLR4 KO mice on NS-HFD presented with improved insulin sensitivity and glucose tolerance compared to WT mice, whereas no differences were observed in the HS-HFD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next assessed whether TLR4 KO influenced exploratory behavior under different dietary conditions using an OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Representative locomotor traces illustrate the differences in exploratory activity between WT and TLR4 KO mice under chow, NS-HFD or HS-HFD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Under chow and HS-HFD, WT and TLR4 KO mice show comparable movement patterns, however under NS-HFD TLR4 KO mice show visibly greater locomotor activity, including frequent entries into the central zone. Additionally, TLR4 KO mice showed a significant increased in total distance travelled, accompanied with a higher percentage of time in the central zone and overall more frequent central zone entries when fed NS-HFD compared to the WT. However, this significance was lost in TLR4 KO fed HS-HFD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). These behavioral differences may suggest that TLR4 KO mice fed NS-HFD present with reduced anxiety-like behavior and enhanced exploratory drive. In contrast, when mice were fed HS-HFD, bot the WT and TLR4 KO mice has comparable locomotor patterns, indicating that the behavioral phenotype associated with TLR4 deficiency is diet specific rather than a generalized effect.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4.0 Discussion","content":"\u003cp\u003eConsistent with previous studies, we found that TLR4 KO protects against diet induced adiposity and insulin resistance under NS-HFD or standard HFD (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). TLR4 KO mice exhibited significantly less weight gain, and improved glucose handling compared to WT controls, this is in line with the established role of TLR4 in mediating lipid-induced inflammation and metabolic dysfunction. These findings reinforce the concept that TLR4 is a key sensor that links dietary fats to systemic metabolic outcomes.\u003c/p\u003e\u003cp\u003eBeyond metabolism, previous studies have assessed the role of TLR4 in cognitive brain function and behavior. Fei et al. demonstrated that TLR4 -/- mice exhibit improved memory abilities and learning, with attenuated fear response (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Similarly, neuronal TLR4 deletion in male mice confers protection against ethanol induced anxiety behaviors (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Additionally, HFD and high cholesterol fed WT C57BL/6J mice were associated with a depressive and anxiety like state mediated by an elevated expression of TLR4 (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Together, these studies point a role for TLR4 in shaping neurobehavioral responses to dietary and environmental stressors.\u003c/p\u003e\u003cp\u003eA novel aspect of our study is the demonstration that this protective effect of TLR4 deficiency is abolished when high sucrose is added into the HFD (HS-HFD). Mice fed HS-HFD gained weight at levels comparable to WT mice and showed no improvement in insulin or glucose sensitivity. This suggests that the detrimental effects of excess sucrose can override the beneficial impacts of TLR4 KO. Importantly, we also observed changes in behavior in TLR4 KO mice fed a NS-HFD or a HS-HFD. Where TLR4 KO mice fed NS-HFD displayed enhanced exploratory activity and reduced anxiety compared to the WT, this phenotype was abolished under HS-HFD. In fact, both mice strains demonstrated restricted central zone exploration, suggesting that not only can the addition of high sucrose to a HFD diminish metabolic protection, but may also promote anxiety-like behaviors in TLR4 KO mice.\u003c/p\u003e\u003cp\u003eOverall, these findings extend prior work by showing that the interaction between dietary fat and sugar fundamentally alters the contribution of innate immune signaling to both metabolic and behavioral outcomes. This highlights the importance of diet composition, and not simply fat alone, that can shape physiology, suggesting that increased sucrose may instigate neurobiological and inflammatory pathways that bypass TLR4. Future studies will be needed to define these exact mechanisms, but our results suggest a close interplay between nutrition, immune signaling, and neurobehavioral health.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eT2D, Type 2 Diabetes\u003c/p\u003e\u003cp\u003eTLR4, Toll-like receptor 4\u003c/p\u003e\u003cp\u003eTLR4 KO, Toll-like receptor 4 knockout\u003c/p\u003e\u003cp\u003eLPS, Lipopolysaccharides\u003c/p\u003e\u003cp\u003eNS-HFD, normal sucrose high fat diet\u003c/p\u003e\u003cp\u003eHS-HFD, high sucrose high fat diet\u003c/p\u003e\u003cp\u003eITT, insulin tolerance test\u003c/p\u003e\u003cp\u003eOGTT, oral glucose tolerance test\u003c/p\u003e\u003cp\u003eOFT, open field test\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this study was obtained from the Animal Care and Use Committee based in Dasman Diabetes Institute\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by funding from the Kuwait Foundation for the Advancement of Science (KFAS), grant no. (RA AM-2020-007; RA-AM 2023-021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGA and RA generated the study, GA curated data and analysis and wrote the first draft. RN performed and assisted in behavioral tests, TJ and SS managed mice cohorts and assisted in data collection, FZ and FA-M reviewed the manuscript, edited and analyzed data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMukherjee S, Kundu R, Vidaković M. Editorial: Metaflammation in obesity and diabetes. 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Neuroscience. 2022;492:1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Y, Zhu S, Xie K, Feng X, Chen L, Wu X, et al. TLR4 in Tph2 neurons modulates anxiety-related behaviors in a sex-dependent manner. Neuropharmacology. 2022;216:109175.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStrekalova T, Evans M, Costa-Nunes J, Bachurin S, Yeritsyan N, Couch Y, et al. Tlr4 upregulation in the brain accompanies depression- and anxiety-like behaviors induced by a high-cholesterol diet. Brain Behav Immun. 2015;48:42\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TLR4KO, Anxiety-like behavior, Obesity, Insulin Resistance, High fat diet with high sucrose","lastPublishedDoi":"10.21203/rs.3.rs-7620609/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7620609/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eObesity and type 2 diabetes (T2D) are accompanied by systemic and neural inflammation that contribute to cognitive and behavioral impairments. Toll-like receptor 4 (TLR4) links nutrient excess to inflammatory signaling and has been implicated in both metabolic dysfunction and neurocognitive decline. While TLR4 deficiency offers protection against high-fat diet (HFD) induced obesity and insulin resistance, the role of dietary sucrose in shaping TLR4 mediated behavioral outcomes remains unclear. We compared wild-type (WT) and TLR4 knockout (KO) mice fed chow, a normal sucrose HFD (NS-HFD), or a high sucrose HFD (HS-HFD) for 13 weeks. TLR4 KO mice on NS-HFD exhibited reduced weight gain, improved insulin sensitivity, and enhanced exploratory behavior with reduced anxiety-like activity in an open field test. However, these protective metabolic and behavioral effects were abolished under HS-HFD, where TLR4 KO and WT mice performed similarly. These findings reveal that high dietary sucrose overrides TLR4 dependent protection, underscoring critical gene and diet interactions in obesity and cognitive dysfunction.\u003c/p\u003e","manuscriptTitle":"TLR4 Deficiency Fails to Protect Against Diet-Induced Behavioral Impairments Under High-Sucrose Feeding","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 11:42:21","doi":"10.21203/rs.3.rs-7620609/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d51aaca4-258f-4e29-a062-e60d6f41da31","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-03T17:09:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-27 11:42:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7620609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7620609","identity":"rs-7620609","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}