Fructose metabolism is associated with anesthesia/surgery induced lactate production

Fructose metabolism is associated with anesthesia/surgery induced lactate production | 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 Research Article Fructose metabolism is associated with anesthesia/surgery induced lactate production Lei Zhang, Jianhui Liu, Zhengjie Miao, Ren Zhou, Hao Wang, Xiang Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4724665/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 Background : Elderly individuals display excessive lactate levels that may contribute to development of cognitive impairment following surgery, including delayed neurocognitive recovery (dNCR). Since the origin of this increased lactate is unknown, here we assessed associations between metabolic pathways and postoperative dNCR. Methods: This study included 43 patients (≥65 years old) who had surgery under general anaesthesia. We also used a mouse model in which 20-month-old mice were exposed under sevoflurane to induce postoperative dNCR. Metabolomics were used to measure metabolites in the serum of patients and brains of mice following anaesthesia/surgery. Isotope labelling and metabolic flux were used to analyse flow and distribution of specific metabolites in metabolic pathways. Results: Among 43 patients, 17 developed dNCR. Metabolomics showed significantly decreased postoperative serum fructose 1-phosphate levels in dNCR compared to non-dNCR patients. Similar results were found in the mouse model. Isotope labelling and metabolic flux experiments in mice showed fructose but not glucose entered glycolysis, increasing lactate levels after anaesthesia/surgery. Administration of intraperitoneal fructose inhibitors to mice effectively inhibited the increased lactate levels and cognitive dysfunction following anaesthesia/surgery. We also found anaesthesia/surgery increased IL-6 levels in mice, and that IL-6 may function upstream in fructose activation. Conclusions: These results suggest that anaesthesia/surgery activates fructose metabolism, producing excessive lactate and ultimately contributing to postoperative cognitive impairment. Fructose metabolism is thus a potential therapeutic target for dNCR. Anesthesiology & Pain Medicine general anesthesia delayed neurocognitive recovery fructose metabolomics lactate Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Points Summary Question: Elderly individuals display increase lactate levels in the brains that may contribute to development of cognitive impairment, however, the origins of the increased lactate remain unknown. Findings: Fructose metabolism, activated by anaesthesia/surgery, leads to increased lactate levels in the brain and is associated with cognitive impairment in aged patients. Meaning: Targeting the fructose metabolic pathway could prevent excessive lactate production and mitigate cognitive impairment in aged patients. Introduction Elderly patients account for 33% of all surgical procedures, yet aging-related changes in these patients lead to poor surgical outcomes. This includes perioperative neurocognitive disorder, which encompasses diverse postoperative cognitive complications including delayed neurocognitive recovery (dNCR) .dNCR is characterized by declining cognitive function within 30 days after anesthesia/surgery, resulting in prolonged hospital stay and even death 1 . Strategies to prevent dNCR could significantly improve surgical health outcomes for the elderly. The elderly brain displays aging-related changes in metabolic processes 2 , which are further affected by exposure to anesthetics. Patients with delirium following hip fracture exhibit impaired glucose utilization in the brain 3 . Our previous results show that clinical concentrations of sevoflurane, the most common inhalation aesthetic, can directly alter glucose metabolism, activate glycolysis, and increase lactate levels in the brains of aged marmosets 4 . Such altered metabolic processes may contribute to postoperative cognitive complications. Indeed, several clinical studies have found that aged patients with postoperative delirium have increased lactate levels in their cerebrospinal fluid 3,5 . Further, sevoflurane exposure in children results in increased lactate concentrations, which are correlated with propensity to exhibit emergence delirium 6 . However, the origins of the increased lactate remain unknown. During surgery, general anesthetics decrease cerebral glucose metabolism 7 , although postoperative cerebrospinal fluid fructose concentrations are higher than preoperative levels in patients undergoing thoracic aortic surgery 8 . Typically, only 1–2% of consumed fructose reaches the brain, but the brain can generate its own fructose. Elevated blood glucose levels alone can increase fructose levels in the brain in healthy individuals 9 . In addition, increasing serum osmolality through dehydration or consuming salty foods 10 or increasing uric acid levels in the brain 11 can increase fructose production. The main difference between glucose and fructose metabolism is that only glucose metabolism can be inhibited by rate-limiting enzymes 12 . This means that fructose can easily enter metabolism, accumulate intermediate products, and convert them into prevalent downstream compounds 13 . Fructose metabolism thus can result in neuroinflammation, brain mitochondrial dysfunction, and oxidative stress, which leads to cognitive impairment 14 . Indeed, the brains of individuals with Alzheimer’s disease show elevated fructose production and metabolism, with 3–5-times higher fructose levels in all areas of the brain compared to controls 15 . Additionally, administration of fructose is associated with increased β-amyloid deposition in animal models of Alzheimer’s disease 15 . This indicates that balance of metabolic pathways and energy sources may be important determinants of brain dysfunction. In this study, we used unbiased metabolomics to assess the brain of aged mice and the serum of elderly patients following anesthesia/surgery. We examined whether activation of fructose or glucose metabolism in the brain results in increased lactate levels and contributes to onset and progression of dNCR. Methods Animal study Mice This study used 20-month-old C57BL/J6 male mice (Beijing Vital River Laboratory Animal Technology Co. Ltd., Beijing, China). The animal protocol was approved by the Institutional Animal Care and Use Committee at Shanghai Ninth People's Hospital (SH9H-2022-A937-1), and relevant ARRIVE guidelines were followed. All mice were first tested in the Y maze to get baseline performance data. Then all mice undergoing surgery, the left carotid artery of mice was exposed under sterile conditions for 30 min under 3% sevoflurane with 40% O 2 at 1 L/min, as done previously 16,17 . Even after the surgery was completed, the mice remained under anesthesia until the duration of total anesthesia lasted for one hour. The anesthetizing chamber was placed in a warm box to maintain mouse rectal temperature at 37 ± 0.5°C. No response to toe pinching was observed during anesthesia. After surgery, all mice received lidocaine ointment. Four days after anesthesia/surgery, mice began training in the Y maze test or were euthanized via decapitation to collect cortical tissues for metabolomics. In total, there are 24 elderly mice. Among them, 10 mice developed dNCR, while 14 mice did not. For metabolomics, we randomly selected 8 cortical tissues samples from a pool of 14 non-dNCR group samples and compared them with 10 cortical tissues samples of dNCR group. To assess the contribution of metabolic pathways in the mouse model, mice were similarly exposed to anesthesia/surgery as above. However, these experiments also included control mice exposed to only 40% O 2 at a flow of 1 L/min. Mice were injected intraperitoneally with 50 mg/kg fructose metabolism inhibitor 2,5-anhydro-D-mannitol (2,5-AM) thirty minutes before anesthesia/surgery. Samples were collected at 6 hours after completion of anesthesia/surgery to measure lactate and IL-6 levels, and mice were tested the Barnes maze at on the 4th day following anesthesia/surgery, by which time the wound has healed, allowing for the initiation of behavioural testing (n = 13; each group). IL-6 knockout mouse experiments were performed in collaboration with Professor Zhongcong Xie at Massachusetts General Hospital. Limited IL-6 knockout mice were available, so experiments were performed with 18-month-old female wildtype (n = 4) and IL-6 knockout (n = 2) mice. Mice underwent abdominal surgery under 1.5–2% isoflurane anesthesia. At 6 h after completion of anesthesia/surgery, mice were euthanized to collect their prefrontal cortex. Isotope labelling and metabolic flux All mice were injected with fructose or glucose intracerebroventricularly. Under anesthesia, the heads of mice were secured in a stereotaxic device, and the epidermis was cut open to locate the Bregma point. From this point, the skull was drilled 0.5 mm towards the tail and 1 mm away from the midline with a grinding drill. An injection needle was inserted at a depth of 2.5 mm to administer 2–2.5 µL of 2 mg/µL fructose (200 mg/kg; n=) or 0.3 mg/µL glucose (30 mg/kg) 18 . Thirty minutes later, control group mice exposed to only 40% O2 at a flow of 1 L/min for one hour. Anesthesia/surgery group mice undergoing surgery, the left carotid artery of mice was exposed under sterile conditions for 30 min under 3% sevoflurane with 40% O 2 at 1 L/min. Even after the surgery was completed, the mice remained under anesthesia until the duration of total anesthesia lasted for one hour. The incision was sutured, and the ipsilateral cortex was removed to detect metabolic flow after 2 h (due to low downstream metabolite labelling at 1 h). We experimented at multiple time points and found that the optimal time was 2 hours post-surgery. Western blotting Mouse cortical tissues were incubated in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, and inhibitors including sodium pyrophosphate, β-glycerophosphate, EDTA, Na 3 VO 4 , and leupeptin). Lysates were separated by SDS–polyacrylamide gel electrophoresis, transferred to PVDF membranes, blocked with 5% non-fat milk, and incubated with primary antibodies against GLUT5 (1:500, 27571-1-AP, Proteintech), MCT1(1:1000, 20139-1-AP, Proteintech), GLUT1 (1:1000, 21829-1-AP, Proteintech), beta-actin (1:20000, 66009-1-Ig, Proteintech), or GAPDH (5174s, Cell Signaling Technology) for 1 h at room temperature. Membranes were visualized with enhanced chemiluminescence substrate (36208ES60, Yeasen Biotechnology, Shanghai, China). Relative target protein levels compared to GAPDH were determined by grey value analysis using Image J. Brain lactate and IL-6 levels Mouse brain lactate levels were detected with a lactate kit (SNM184, Biolab Co., Ltd. China) according to manufacturer instructions. Optical density of wells was measured with a fluorescence plate reader (Medical Device, San Jose, CA, USA) at 530 nm. Mouse brain IL-6 levels were detected with an ELISA kit (abs552203, Absin Bioscience, Shanghai China) according to manufacturer instructions. Optical density of wells was measured with a fluorescence plate reader (Medical Device) at 450 nm. Standard curves were generated using R graphing software (4.0.5, https://www.r-project.org ). Barnes maze test Mice were tested in the Barnes maze to assess spatial learning and memory 19 . Phase I – Habituation : Mice were habituated to the environment for at least 30 min. Light intensity was set to ~ 1000 Lux. Speakers were activated, and aversive noise was set to 90 dB. Mice were situated in the centre of a circular platform with 20 evenly spaced holes. Mice were guided with a black lid to the escape hole and remained there for 2 min. Mice rested for at least 30 min. The same process was repeated daily. Phase II - Training : Mice were placed in the centre and covered with a black lid for 15 s. The black lid was removed while turning on the aversive noise. The experiment lasted 3 min. If the mouse successfully entered the escape hole, the aversive noise stopped and the experiment ended. The mouse was allowed to stay in the escape tunnel for 15 s before removal. If the mouse failed to find the escape hole, it was guided there with a black lid and allowed to stay there for 15 s. Consistent with a previous study 16 , mice began training 4 days after anesthesia/surgery, and spatial memory tests were conducted 5 days after training. The EthoVision-XT video tracking system (Noldus, Wageningen, and the Netherlands) was used to monitor mice in the maze. dNCR was defined as postoperative Y maze scores > 5% decreased compared to preoperative scores. Clinical study Study enrolment This prospective observational cohort study was conducted at Shanghai Tongji Hospital at Tongji University in 2021–2022. After receiving approval from the ethics committee (2021 − 111) as well as written informed consent, we recruited 43 patients. Preoperative inclusion criteria were: 1) ≥ 60 years old; 2) undergoing optional spinal surgery; 3) American Society of Anesthesiologists physical status of I–III; and 4) Mini-Mental State Examination score higher than the minimum score for the patient’s education level [≥ 20 for primary school (≤ 6 years of education), or ≥ 23 for middle school and higher (> 6 years of education)]. Preoperative exclusion criteria were: 1) hearing, vision, or speech barriers that prevented cooperation with perioperative neuropsychological tests; 2) nervous system disease or mental illness; 3) serious systemic diseases or severe postoperative complications (e.g., bleeding, cerebral infarction, pulmonary infection); or 4) metal implants, artificial cochlear implants, cardiac pacemakers, and other implanted devices. This study adhered to STROBE guidelines. The study protocol was registered with chictr.org.cn (ChiCTR2200057080). Anesthesia, surgery, and serum sample collection All patients received general anesthesia under tracheal intubation. The induction drugs used were sufentanil (20–30 µg), etomidate (0.3–0.4 mg/kg), and cis-atracurium (0.2 mg/kg). A preoperative antiemetic (e.g., ramosetron) was used as required. Intraoperative intravenous inhalation anesthesia used was sevoflurane (1–1.5%) combined with propofol (2–6 mg/kg/h) and remifentanil (0.05–1 µg/kg/min). Sufentanil and cis-atracurium were used intermittently to deepen anesthesia and relax muscles as needed. Patient anesthesia control was according to bleeding volume, urine volume, and arterial blood pressure. Timely replenishment of crystalloid (lactated Ringer's) and colloid (succinyl gelatin) intravenous solutions was maintained to monitor blood cell production. We used a Fabius GS premium anesthetic workstation (Dräger, Lübeck, Germany) for general anesthesia, a multifunctional monitor (GE Carescape B850, Boston, MA, USA) to measure the patient’s vital signs, and anesthesia depth monitor (ConView YY-106, Pearlcare, Zhejiang, China) to determine depth of anesthesia. Intraoperative blood gas analysis (GEM Premier 3500, Werfen, Spain) was used to monitor blood oxygen and adjust water–electrolyte balance. All participants received standardized perioperative care, including postoperative pain management using sufentanil (100 µg/48 h)/hydromorphone (8 mg/48 h) patient-controlled analgesia. We collected 5 mL of venous blood from the internal jugular vein of participants before and immediately after surgery. Separation gel and coagulant were added to blood tubes (20231280, Shanghai Orsin Medical Technology Co. Ltd., Shanghai, China). Samples were incubated at 25°C for 30 min and centrifuged at 3000 rpm for 15 min at 4°C. Serum supernatant was collected and stored at -80°C. dNCR measurement Trained clinical researchers assessed participants for cognition change before surgery and prior to being discharged or at 7 days post-surgery using the Digit Symbol Substitution Test, Trail Making Test A, Judgment of Line Orientation Test, Stroop Color Word Test, and Auditory Verbal Learning Test, with a 3 min gap between intervals. At 30 days post-surgery, the Abbreviated Mental Test Score was assessed over the phone. A decrease of one SD in both evaluation scales after surgery was considered dNCR 20 . Patients who did not develop dNCR constituted the control group. Liquid chromatography–tandem mass spectrometry Consistent with a previous study 21 , 50 µL of patient serum and 300 µL of methanol were mixed by vortexing for 10 min at 4°C and 2000 rpm. After centrifugation at 4°C for 10 min at 12,000 rpm, 300 µL of supernatant were transferred to a new tube and dried using a stream of nitrogen. Dry residue was reconstituted in 50 µL of 2% acetonitrile, mixed by vortexing at 4°C for 10 min at 2000 rpm, and centrifuged at 4°C for 10 min at 12,000 rpm. The supernatant was transferred to an autosampler vial. Sample extracts were analysed by reversed-phase chromatography using a mobile phase of water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid) at a constant rate of 0.20 mL/min. The injection volume was 1 µL, and the temperature of the autosampler was 40°С. Chromatographic separation was achieved on an Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm, Waters Corp., Milford, MA, USA) on a Vanquish system (Thermo Fisher Scientific, Waltham, MA, USA). A TSQ Altis mass spectrometer (Thermo Fisher Scientific) with electrospray ionization source was operated in multiple reaction monitoring mode for mass data acquisition. The parameters of the heated electrospray ionization source were: sheath gas at 40 arbitrary units, aux gas at 10 arbitrary units, spray voltage at 3.5 kV (+)/2.5 kV (-), ion transfer tube temperature at 320°C, and vaporizer temperature at 325°C. Optimized mass spectrometer conditions were used for quantitative analysis. Statistical analysis Clinical data Data are expressed as mean ± SD if normally distributed; otherwise, they are expressed as median and 25th and 75th percentiles. Differences between groups were analysed using student’s t -test, Wilcoxon test, or Fisher's exact test as appropriate. Mean and SD of patients’ preoperative and postoperative scale scores were calculated. Metabolomics data Widely targeted metabolomics data included 543 metabolites from 100 samples. The online analytical tool MetaboAnalyst 5.0 ( https://www.metaboanalyst.ca/ ) was used to perform multivariate (multidimensional) statistical analysis. Multivariate statistical methods such as principal component analysis, partial least squares discrimination analysis, and orthogonal partial least squares discrimination analysis were used for comparative analysis of the whole metabolic spectrum and metabolite screening. Data were normalized before multivariate statistical modelling. Differential metabolites were selected using criteria of variable importance in projection (VIP) > 1.0 and student's t-test p < 0.05. Results Baseline participant characteristics The only baseline metrics that significantly differed between dNCR and non-dNCR groups were blood glucose ( p = 0.046) and blood creatinine ( p = 0.052) (Table 1). dNCR patients have significantly decreased postoperative serum fructose 1-phosphate levels Among 43 TJ participants, 17 patients developed dNCR. We applied orthogonal partial least squares discrimination analysis to postoperative serum metabolomics data for 543 metabolites between dNCR and non-dNCR groups (Fig. 1 A). Based on VIP values, we ranked the top differential metabolites between groups (Fig. 1 B). Postoperative serum levels of fructose 1-phosphate—which can be directly converted into lactate—were significantly decreased in dNCR compared to non-dNCR groups (Fig. 1 C,D). dNCR mice have significantly decreased postoperative brain fructose 1-phosphate levels We investigated whether fructose 1-phosphate levels also were associated with dNCR in our mouse model. At four days post-anesthesia/surgery, mice were tested in the Y maze to assess postoperative cognitive impairment (Fig. 2 A). Among 24 mice, 10 developed dNCR (Fig. 2 B). We applied orthogonal partial least squares discrimination analysis to postoperative brain metabolomics data for 543 metabolites in the brain of mice in dNCR (n = 10) and non-dNCR groups (n = 8, randomly chosen) (Fig. 2 C). We identified differential metabolites between dNCR and non-dNCR groups (Fig. 2 D), including significantly decreased fructose 1-phosphate levels in dNCR compared to non-dNCR mice (Fig. 2 E,F). Decreased metabolites can be attributed to either metabolic decreased yield or increased consumption. A member of the facilitative glucose transporter (GLUT) family, GLUT5, is responsible for passive membrane transport of fructose 22 . GLUT5 is the only transporter specific for fructose and cannot transport glucose or galactose 23 . Interestingly, GLUT5 levels were increased in dNCR compared to non-dNCR groups (Fig. 2 G,H). Elevated lactate levels are associated with activation of fructose metabolism We investigated whether the increased lactate levels in mice were due to fructose metabolism or glucose metabolism. Fructose can enter glycolysis through two pathways: the fructokinase pathway, or the hexokinase pathway 24 (Fig. 3 A). At 2 h after anesthesia/surgery, mouse brains had increased levels of downstream fructose decomposition products (e.g., fructose 6-phosphate, fructose 1-phosphate) (Fig. 3 B,C) as well as increased lactate production (Fig. 3 D). Glucose also can enter glycolysis directly (Fig. 3 E), although altered glucose to lactate metabolism was not observed following anesthesia/surgery compared to control mice (Fig. 3 F–H). We then used intraperitoneal injection of fructose metabolism inhibitor 2,5-anhydro-D-mannitol (2,5-AM) 25 to determine the effect of fructose metabolism on anesthesia/surgery-induced learning and memory dysfunction (Fig. 4 A). Administration of 2,5-AM prevented the increased lactate levels in the brains of elderly mice following anesthesia/surgery (Fig. 4 B). While mice exposed to anesthesia/surgery had longer latency to find the target and spent less time in the entry zone of the Barnes maze compared to control mice, administration of 2,5-AM also prevented these deficits (Fig. 4 C,D). Anesthesia/surgery-induced elevation of IL-6 may be related to fructose metabolism in the brains of elderly mice A high-fructose diet increases neuroinflammation 14 , and neuroinflammation contributes to cognitive impairment in rodents following surgery under general anesthesia 26 . In addition, inflammation also activates fructose metabolism and especially IL-6 27–29 . IL-6 is both necessary and sufficient to produce cognitive decline in mice 26,30 and patients 1 . To clarify the upstream and downstream connections between neuroinflammation and fructose metabolism, we injected the fructose metabolism inhibitor 2,5-AM into the abdominal cavity of elderly mice before anesthesia/surgery. Previous research shows that abdominal surgery can lead to postoperative cognitive impairment in mice 31 . While anesthesia/surgery significantly increased IL-6 levels in the brains of mice, fructose metabolism inhibitors only had a slight salvage effect on elevated IL-6 levels following anesthesia/surgery (Fig. 5 A). However, IL-6 knockout transgenic mice exhibited decreased expression of Glut5 in the brain after anesthesia/surgery compared to wildtype mice (Fig. 5 B). Surprisingly, expression of glucose transmembrane transport protein GLUT1, which serves as a crucial gating protein to control passage of glucose into and out of tissues, was unaffected by IL-6 knockout. In addition, lactate exchange is facilitated by MCT1, which handles uptake of exogenous lactate by glycolysis 32 . IL-6 knockout transgenic mice exhibited decreased Mct1 expression in the brain after anesthesia/surgery compared to wildtype mice (Fig. 5 B). Discussion Our study shows that elderly patients who underwent anesthesia/surgery and developed dNCR had decreased fructose 1-phosphate levels in their blood compared to those who did not develop dNCR. We found similar results in our mouse model. Further, mice in the dNCR group had increased Glut5 expression compared to the non-dNCR group, suggesting anesthesia/surgery activates fructose metabolism. Metabolic flux experiments showed that activation of fructose metabolism increased glycolysis and lactate production, and that fructose inhibitors could inhibit the anesthesia/surgery-induced increase in lactate. Further, administering fructose inhibitors to mice helped prevent the postoperative cognitive decline induced by anesthesia/surgery. In addition, our results indicate that IL-6 could be the upstream mechanism to activate fructose metabolism. Taken together, these data suggest that anesthesia/surgery activates fructose metabolism in the brain to increase lactate levels, contributing to dNCR. Fructose can be converted into fructose 6-phosphate by hexokinase and then enter glycolysis. Phosphate fructose kinase serves as the rate-limiting enzyme in this step. However, fructose also can be converted by fructose kinase (ketohexokinase) into fructose 1-phosphate, which also participates in glycolysis. Notably, there is no rate-limiting enzyme involved in this pathway. Fructose is more readily converted to lactate than glucose. We determined that anesthesia/surgery triggered fructose metabolism and increased fructose 1-phosphate levels, causing fructose to enter glycolysis and produce lactate. Fructose 1-phosphate and fructose metabolism thus may serve as potential therapeutic targets for dNCR. Fructose activation is potentially neurotoxic through various mechanisms. First, conversion of fructose into fat is a simple process 33 . The liver plays a crucial role in metabolizing fructose, but excessive consumption can result in low high-density lipoprotein cholesterol levels and high hepatic inflammation 34 . In addition, dysfunctional lipid metabolism in astrocytes is crucial in dNCR development 35 . Second, high fructose consumption disrupts brain mitochondrial function and may contribute to neurological disorders. In both short-term 36 and long-term 37 experiments, high fructose consumption results in significantly decreased levels of major mitochondrial functional markers in the brain. High fructose consumption changes the brain’s unfolded protein response and macroautophagic machinery, promoting build-up of aggregates of β-amyloid 1–42 (Aβ42), tau-p-S199, and tau-p-S404, which are indicators of neurodegeneration 38 . Third, activation of fructose metabolism can result in insulin resistance 12 . Clinical postoperative cognitive dysfunction can be effectively predicted by preoperative insulin resistance 39,40 or postoperative insulin resistance 3 indicating that targeted prevention and treatment strategies for insulin resistance may be effective interventions for at-risk patient Despite our significant findings, this study is limited in that our experimental design assessed mice at 4 days after anesthesia/surgery, but the increase in fructose 1-phosphate may have occurred earlier. A previous clinical study shows that fructose levels surge in cerebrospinal fluid at 2 days post-anesthesia/surgery 8 . Yet that study was limited by its sample size, so perioperative cognitive tests were not performed and the association between fructose metabolism and perioperative neurocognitive disorder was not clear. Yet we believe that study and our study are complementary, showing correlation between fructose metabolism activated by anesthesia/surgery and postoperative dNCR. In addition, we used IL-6 knockout transgenic mice to indicate that the increased IL-6 caused by anesthesia/surgery may trigger fructose metabolism. Unfortunately, we only had two 18-month-old female elderly IL-6 knockout transgenic mice, so they cannot be statistically analysed. Nevertheless, given that elderly IL-6 knockout transgenic mice exhibited significantly decreased Glut5 expression in the brain after anesthesia/surgery, it is feasible that neuroinflammation acts as an upstream mechanism to activate fructose metabolism. That study was limited by it did not pinpoint the origin of fructose. Typically derived from food, fructose is primarily stored in the intestines and liver. Moreover, clinical patients often undergo fasting and hydration before anesthesia. It has been discovered that the brain generates endogenous fructose, leading to speculation that anesthesia and surgery may trigger the conversion of other substances like glutamine into fructose, thus impacting fructose metabolism. Future research may focus on uncovering the source of fructose. In conclusion, our results indicate that anesthesia/surgery-induced neuroinflammation can reprogram glucose metabolism, ultimately leading to dNCR. This reprogramming may be driven by increased IL-6 levels in the brain to activate fructose metabolism. Downstream metabolites of fructose then enter glycolysis, producing excessive lactate and ultimately contributing to postoperative cognitive impairment. Thus, fructose metabolism may be a potential treatment for postoperative neurocognitive disorder. Declarations Funding : This research was supported by the National Natural Science Foundation of China (82171173) and Clinical Research Program of 9 th People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine (JYLJ202221, JYLJ202304). Declaration of interests: The authors declare that they have no conflict of interest. The study protocol was registered with chictr.org.cn (ChiCTR2200057080) https://www.chictr.org.cn/showproj.html?proj=152662 Justification of authorship : LZ: conceptualization, investigation, writing – reviewing and editing, funding acquisition. JH L, JH L, JY Z: patient recruitment, data collection. RZ: software, validation. ZJ M, HW, and XL: methodology. JY: data curation. HJ: conceptualization, writing – reviewing and editing. Data availability The datasets generated and/or analysed during this study are not publicly available but are available from the corresponding author on reasonable request. Acknowledgement : This research was supported by the National Natural Science Foundation of China (82171173) and Clinical Research Program of 9 th People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine (JYLJ202221, JYLJ202304). References Li Y, Chen D, Wang H, et al. Intravenous versus Volatile Anesthetic Effects on Postoperative Cognition in Elderly Patients Undergoing Laparoscopic Abdominal Surgery. Anesthesiology . Mar 1 2021;134(3):381-394. doi:10.1097/ALN.0000000000003680 Mahajan UV, Varma VR, Griswold ME, et al. 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Mar 5 2018;8(5):e2744. doi:10.21769/bioprotoc.2744 Rasmussen LS, Larsen K, Houx P, et al. The assessment of postoperative cognitive function. Acta Anaesthesiol Scand . Mar 2001;45(3):275-89. doi:10.1034/j.1399-6576.2001.045003275.x Yuan M, Breitkopf SB, Yang X, Asara JM. A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nat Protoc . Apr 12 2012;7(5):872-81. doi:10.1038/nprot.2012.024 Burant CF, Takeda J, Brot-Laroche E, Bell GI, Davidson NO. Fructose transporter in human spermatozoa and small intestine is GLUT5. J Biol Chem . Jul 25 1992;267(21):14523-6. Douard V, Ferraris RP. Regulation of the fructose transporter GLUT5 in health and disease. Am J Physiol Endocrinol Metab . Aug 2008;295(2):E227-37. doi:10.1152/ajpendo.90245.2008 Nakagawa T, Lanaspa MA, Millan IS, et al. Fructose contributes to the Warburg effect for cancer growth. Cancer Metab . 2020;8:16. doi:10.1186/s40170-020-00222-9 Tordoff MG, Rafka R, DiNovi MJ, Friedman MI. 2,5-anhydro-D-mannitol: a fructose analogue that increases food intake in rats. Am J Physiol . Jan 1988;254(1 Pt 2):R150-3. doi:10.1152/ajpregu.1988.254.1.R150 Dong Y, Xu Z, Huang L, Zhang Y, Xie Z. Peripheral surgical wounding may induce cognitive impairment through interlukin-6-dependent mechanisms in aged mice. Med Gas Res . Oct-Dec 2016;6(4):180-186. doi:10.4103/2045-9912.196899 Huang X, Fang J, Lai W, et al. IL-6/STAT3 Axis Activates Glut5 to Regulate Fructose Metabolism and Tumorigenesis. Int J Biol Sci . 2022;18(9):3668-3675. doi:10.7150/ijbs.68990 Zhou J, Yang J, Wang YM, et al. IL-6/STAT3 signaling activation exacerbates high fructose-induced podocyte hypertrophy by ketohexokinase-A-mediated tristetraprolin down-regulation. Cell Signal . Oct 2021;86:110082. doi:10.1016/j.cellsig.2021.110082 Yahia H, Hassan A, El-Ansary MR, Al-Shorbagy MY, El-Yamany MF. IL-6/STAT3 and adipokine modulation using tocilizumab in rats with fructose-induced metabolic syndrome. Naunyn Schmiedebergs Arch Pharmacol . Dec 2020;393(12):2279-2292. doi:10.1007/s00210-020-01940-z Hu J, Feng X, Valdearcos M, et al. Interleukin-6 is both necessary and sufficient to produce perioperative neurocognitive disorder in mice. Br J Anaesth . Mar 2018;120(3):537-545. doi:10.1016/j.bja.2017.11.096 Liufu N, Liu L, Shen S, et al. Anesthesia and surgery induce age-dependent changes in behaviors and microbiota. Aging (Albany NY) . Jan 24 2020;12(2):1965-1986. doi:10.18632/aging.102736 Bonen A. The expression of lactate transporters (MCT1 and MCT4) in heart and muscle. Eur J Appl Physiol . Nov 2001;86(1):6-11. doi:10.1007/s004210100516 Herman MA, Birnbaum MJ. Molecular aspects of fructose metabolism and metabolic disease. Cell Metab . Dec 7 2021;33(12):2329-2354. doi:10.1016/j.cmet.2021.09.010 Abdelmalek MF, Suzuki A, Guy C, et al. Increased fructose consumption is associated with fibrosis severity in patients with nonalcoholic fatty liver disease. Hepatology . Jun 2010;51(6):1961-71. doi:10.1002/hep.23535 Zhang L, Wang X, Yu W, et al. CB2R Activation Regulates TFEB-Mediated Autophagy and Affects Lipid Metabolism and Inflammation of Astrocytes in POCD. Front Immunol . 2022;13:836494. doi:10.3389/fimmu.2022.836494 Jiménez-Maldonado A, Ying Z, Byun HR, Gomez-Pinilla F. Short-term fructose ingestion affects the brain independently from establishment of metabolic syndrome. Biochim Biophys Acta Mol Basis Dis . Jan 2018;1864(1):24-33. doi:10.1016/j.bbadis.2017.10.012 Cigliano L, Spagnuolo MS, Crescenzo R, et al. Short-Term Fructose Feeding Induces Inflammation and Oxidative Stress in the Hippocampus of Young and Adult Rats. Mol Neurobiol . Apr 2018;55(4):2869-2883. doi:10.1007/s12035-017-0518-2 Bermejo-Millo JC, Guimarães MRM, de Luxán-Delgado B, et al. High-Fructose Consumption Impairs the Redox System and Protein Quality Control in the Brain of Syrian Hamsters: Therapeutic Effects of Melatonin. Mol Neurobiol . Oct 2018;55(10):7973-7986. doi:10.1007/s12035-018-0967-2 He X, Long G, Quan C, Zhang B, Chen J, Ouyang W. Insulin Resistance Predicts Postoperative Cognitive Dysfunction in Elderly Gastrointestinal Patients. Front Aging Neurosci . 2019;11:197. doi:10.3389/fnagi.2019.00197 Tang N, Jiang R, Wang X, et al. Insulin resistance plays a potential role in postoperative cognitive dysfunction in patients following cardiac valve surgery. Brain Res . Feb 15 2017;1657:377-382. doi:10.1016/j.brainres.2016.12.027 Table Table 1 is available in the Supplementary Files section Additional Declarations The authors declare no competing interests. Supplementary Files Table1.docx Baseline characteristics for all participants 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. 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-4724665","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":325717312,"identity":"53e2a6af-8ea8-411d-9acb-77a474401beb","order_by":0,"name":"Lei Zhang","email":"","orcid":"","institution":"Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhang","suffix":""},{"id":325717313,"identity":"c9896800-d548-44b8-9b5e-be9c018f8034","order_by":1,"name":"Jianhui Liu","email":"","orcid":"","institution":"Tongji Hospital, School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Jianhui","middleName":"","lastName":"Liu","suffix":""},{"id":325718978,"identity":"46a48b27-f04e-4155-b4b7-33f1c5ef473d","order_by":2,"name":"Zhengjie Miao","email":"","orcid":"","institution":"Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhengjie","middleName":"","lastName":"Miao","suffix":""},{"id":325718979,"identity":"bcef0b74-24b2-4343-b96a-d3cf70b8b164","order_by":3,"name":"Ren Zhou","email":"","orcid":"","institution":"Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ren","middleName":"","lastName":"Zhou","suffix":""},{"id":325718980,"identity":"b7abe15f-6adb-4860-8423-5ef92060bdcc","order_by":4,"name":"Hao Wang","email":"","orcid":"","institution":"Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Wang","suffix":""},{"id":325718981,"identity":"ac7d2671-97cf-491c-9eb5-b9aa53b817a3","order_by":5,"name":"Xiang Li","email":"","orcid":"","institution":"Critical Care and Pain Medicine, Massachusetts General Hospital, Harvard Medical School","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Li","suffix":""},{"id":325718982,"identity":"66c8a98c-76e4-4c2f-ae61-cfa604781ae9","order_by":6,"name":"Jiehui Liu","email":"","orcid":"","institution":"Tongji Hospital, School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Jiehui","middleName":"","lastName":"Liu","suffix":""},{"id":325718983,"identity":"dcabda56-587d-49c9-9db7-ba59f38c1b78","order_by":7,"name":"Jingya Zhang","email":"","orcid":"","institution":"Tongji Hospital, School of Medicine, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Jingya","middleName":"","lastName":"Zhang","suffix":""},{"id":325718984,"identity":"6695833c-3bab-436d-a550-e3b56585cfec","order_by":8,"name":"Jia Yan","email":"","orcid":"","institution":"Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Yan","suffix":""},{"id":325718985,"identity":"0185ccb4-0638-43f1-8bb6-3eba4e8a70a3","order_by":9,"name":"Hong Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYBACxmYQ0QBiHgSTDPzMzIcfkKZFsp0tzYCwVQ1IHIPzPAoS+FQztzM/e/h1h02evOPhNmneNrs848M8DAYMNTbRuB3GZm4seyat2PDAQZCW5GKzw7wHHjAcS8ttwKmFwUxasu1w4sYGsBbmxG2H+RIMGBsO49HC/g1ZS33i5mYeAwn8WnjMJD8CtcxnAGs5nLiBmbCWMmnGtrTEDQwHmy3nnDueOOMwMJAT8PjFsP/4NsmfbTaJ82ccf3jjTVl1Yn//4cMPPtTY4NYClGDmATIMbhxgYOKBCSfgUA4C8iDH/QAx+hsgjFEwCkbBKBgF6AAAZapgsNdpH/kAAAAASUVORK5CYII=","orcid":"","institution":"Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Hong","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2024-07-11 14:07:52","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-4724665/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4724665/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60341770,"identity":"ebcd60f0-d6c5-448a-a305-51735ecdf6be","added_by":"auto","created_at":"2024-07-15 18:45:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":345113,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of serum metabolites in elderly human patients. A: Orthogonal partial least squares discrimination analysis of postoperative serum between delayed neurocognitive recovery (dNCR) and non-dNCR groups, T score [1] (2.7%) and orthogonal T score [1] (11.8%). B: Top 15 differential metabolites in postoperative serum between dNCR and non-dNCR groups [variable importance in projection (VIP) \u0026gt; 1]. C: Volcano plot of metabolites in postoperative serum from dNCR and non-dNCR groups (fold-change \u0026gt; 1.2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). D: Serum fructose 1-phosphate concentrations in dNCR and non-dNCR groups determined by metabolomics (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). D.\u003cem\u003et-\u003c/em\u003etest with SD.\u003c/p\u003e","description":"","filename":"FIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-4724665/v1/35af290cd611a7f92fdb02f4.png"},{"id":60341757,"identity":"c914d429-16af-4e94-98be-4144a3b96cce","added_by":"auto","created_at":"2024-07-15 18:45:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":585679,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of serum metabolites in elderly mice. A: Schematic for determination of delayed neurocognitive recovery (dNCR) in the mouse model following anesthesia/surgery (A/S). All mice received surgery under sevoflurane anesthesia. Y maze test was used to evaluate their behaviour before and 4 days after the procedure. Elderly mice with postoperative Y maze scores decreased by \u0026gt;5% compared to preoperative scores were considered to have dNCR. B: Out of 24 elderly male mice, 10 developed dNCR while 14 did not (***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001). C: Orthogonal partial least squares discrimination analysis of postoperative metabolites in the brains of mice between dNCR and non-dNCR groups, T score [1] (9.8%) and orthogonal T score [1] (31.7%). D: Top 20 differential metabolites in brains of mice between dNCR and non-dNCR groups [variable importance in projection (VIP) \u0026gt; 1]. E: Volcano plot of postoperative metabolites in the brains of mice in dNCR and non-dNCR groups (fold-change \u0026gt; 1.2, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05). F: Fructose 1-phosphate levels in the brains of mice in dNCR and non-dNCR groups (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). G: Western blot of GLUT5 protein levels in dNCR and non-dNCR groups. Actin is a loading control. H: Statistical analysis of \u003cem\u003eGlut5\u003c/em\u003e expression in dNCR and non-dNCR groups (**\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01). F.\u003cem\u003et\u003c/em\u003e-test with SD. H.\u003cem\u003et\u003c/em\u003e-test with SD.\u003c/p\u003e","description":"","filename":"FIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-4724665/v1/49721ac905588bec9abba21f.png"},{"id":60341762,"identity":"cb363957-01c2-4e64-a487-8b2758b6992f","added_by":"auto","created_at":"2024-07-15 18:45:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":358596,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of metabolic pathways in elderly mice. A: Schematic for entry of fructose metabolism into glycolysis. B–D: The lateral ventricle of the brains of aged mice exposed to control (Ctrl) or anesthesia/surgery (A/S) conditions was injected with 200 mg/kg of isotopically labelled fructose, and fructose 6-phosphate (B), fructose 1-phosphate (C), and lactate (D) levels were measured after 2 h. E: Schematic for entry of glucose metabolism into glycolysis. F–H: The lateral ventricle of the brains of aged mice exposed to control (Ctrl) or anesthesia/surgery (A/S) conditions was injected with 30 mg/kg of isotopically labelled glucose, and glucose 1-phosphate (F), glucose 6-phosphate (G), and lactate (H) levels were measured after 2 h. M0, M1, M2. M\u003cem\u003en\u003c/em\u003e refer to isotopologues containing \u003cem\u003en\u003c/em\u003e heavy atoms in a molecule. B, C, D, F, G, H: One-wayanova. Post hoc test (turkey’s multiple comparisons test with SD).\u003c/p\u003e","description":"","filename":"FIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-4724665/v1/b25cccb0834bf1ec9e4e6b87.png"},{"id":60341761,"identity":"7333ac29-3a2f-4750-bde3-e9c90f2fc2c3","added_by":"auto","created_at":"2024-07-15 18:45:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":418901,"visible":true,"origin":"","legend":"\u003cp\u003eFructose metabolism inhibitor mitigates postoperative cognitive impairment in aged mice exposed to anesthesia/surgery. A: Schematic of experimental design for aged mice (20 months old). Mice in the control (Ctrl) group were exposed to 40% oxygen, and the anesthesia/surgery (A/S) group underwent surgery. B: Lactate levels in the brains of elderly mice following control or anesthesia/surgery conditions and intraperitoneal injection of fructose metabolism inhibitor 2,5-anhydro-D-mannitol (2,5-AM; 50 mg/kg) (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05; ****\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001). C: Latency to identify target in Barnes maze test training sessions for mice exposed to control or anesthesia/surgery conditions as well as treatment with 2,5-AM. **\u003cem\u003ep\u003c/em\u003e = 0.0029, F (1,112) = 9.262 for A/S vs control group. **\u003cem\u003ep\u003c/em\u003e= 0.0032, F (1,112) = 9.065 for (A/S) + 2,5-AM vs A/S group. D: Time in the entry zone in Barnes maze test for mice exposed to control or anesthesia/surgery conditions as well as treatment with 2,5-AM (***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001). B.one-way anova. Post hoc test(turkey’s multiple comparisons test with SEM)，C. two-way ANOVA with repeated-measurement analysis showed no significant interaction between treatment，Post hoc test(sidak's multiple comparisons test with SEM).\u003c/p\u003e","description":"","filename":"FIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-4724665/v1/64998d63f33d9eae2056798f.png"},{"id":60341758,"identity":"3019dad1-5a55-4366-b529-1c5a233fdc7b","added_by":"auto","created_at":"2024-07-15 18:45:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":411694,"visible":true,"origin":"","legend":"\u003cp\u003eAnesthesia/surgery-induced elevation of IL-6 in the brains of elderly mice. A: IL-6 levels in the brains of mice exposed to control or anesthesia/surgery (A/S) conditions as well as treatment with fructose metabolism inhibitor 2,5-anhydro-D-mannitol (2,5-AM; 50 mg/kg). B: Western blots of GLUT5, MCT1, and GLUT1 levels in the brains of IL-6 knockout transgenic and wild-type mice after anesthesia/surgery. A.one-way anova, Post hoc test (turkeys multiple comparisons test with SEM).\u003c/p\u003e","description":"","filename":"FIG5.png","url":"https://assets-eu.researchsquare.com/files/rs-4724665/v1/e9e3524b9e551c1323f8bb25.png"},{"id":60342414,"identity":"3a71ea9f-02a7-4276-8e1c-4a5e37ab79fc","added_by":"auto","created_at":"2024-07-15 19:01:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2528339,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4724665/v1/2ab2cce8-cc95-4379-83a4-7e20b23a19a4.pdf"},{"id":60341756,"identity":"064ba6f8-dd5b-4a3e-be55-b9e431afb9c9","added_by":"auto","created_at":"2024-07-15 18:45:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBaseline characteristics for all participants\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4724665/v1/05b355779d3961c1db4d689c.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eFructose metabolism is associated with anesthesia/surgery induced lactate production\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Key Points Summary ","content":"\u003cp\u003eQuestion: Elderly individuals display increase lactate levels in the brains that may contribute to development of cognitive impairment, however, the origins of the increased lactate remain unknown.\u003c/p\u003e\n\u003cp\u003eFindings: Fructose metabolism, activated by anaesthesia/surgery, leads to increased lactate levels in the brain and is associated with cognitive impairment in aged patients.\u003c/p\u003e\n\u003cp\u003eMeaning: Targeting the fructose metabolic pathway could prevent excessive lactate production and mitigate cognitive impairment in aged patients.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eElderly patients account for 33% of all surgical procedures, yet aging-related changes in these patients lead to poor surgical outcomes. This includes perioperative neurocognitive disorder, which encompasses diverse postoperative cognitive complications including delayed neurocognitive recovery (dNCR) .dNCR is characterized by declining cognitive function within 30 days after anesthesia/surgery, resulting in prolonged hospital stay and even death \u003csup\u003e1\u003c/sup\u003e. Strategies to prevent dNCR could significantly improve surgical health outcomes for the elderly.\u003c/p\u003e \u003cp\u003eThe elderly brain displays aging-related changes in metabolic processes \u003csup\u003e2\u003c/sup\u003e, which are further affected by exposure to anesthetics. Patients with delirium following hip fracture exhibit impaired glucose utilization in the brain\u003csup\u003e3\u003c/sup\u003e. Our previous results show that clinical concentrations of sevoflurane, the most common inhalation aesthetic, can directly alter glucose metabolism, activate glycolysis, and increase lactate levels in the brains of aged marmosets \u003csup\u003e4\u003c/sup\u003e. Such altered metabolic processes may contribute to postoperative cognitive complications. Indeed, several clinical studies have found that aged patients with postoperative delirium have increased lactate levels in their cerebrospinal fluid \u003csup\u003e3,5\u003c/sup\u003e. Further, sevoflurane exposure in children results in increased lactate concentrations, which are correlated with propensity to exhibit emergence delirium \u003csup\u003e6\u003c/sup\u003e. However, the origins of the increased lactate remain unknown.\u003c/p\u003e \u003cp\u003eDuring surgery, general anesthetics decrease cerebral glucose metabolism \u003csup\u003e7\u003c/sup\u003e, although postoperative cerebrospinal fluid fructose concentrations are higher than preoperative levels in patients undergoing thoracic aortic surgery \u003csup\u003e8\u003c/sup\u003e. Typically, only 1\u0026ndash;2% of consumed fructose reaches the brain, but the brain can generate its own fructose. Elevated blood glucose levels alone can increase fructose levels in the brain in healthy individuals \u003csup\u003e9\u003c/sup\u003e. In addition, increasing serum osmolality through dehydration or consuming salty foods \u003csup\u003e10\u003c/sup\u003e or increasing uric acid levels in the brain \u003csup\u003e11\u003c/sup\u003e can increase fructose production.\u003c/p\u003e \u003cp\u003eThe main difference between glucose and fructose metabolism is that only glucose metabolism can be inhibited by rate-limiting enzymes \u003csup\u003e12\u003c/sup\u003e. This means that fructose can easily enter metabolism, accumulate intermediate products, and convert them into prevalent downstream compounds \u003csup\u003e13\u003c/sup\u003e. Fructose metabolism thus can result in neuroinflammation, brain mitochondrial dysfunction, and oxidative stress, which leads to cognitive impairment \u003csup\u003e14\u003c/sup\u003e. Indeed, the brains of individuals with Alzheimer\u0026rsquo;s disease show elevated fructose production and metabolism, with 3\u0026ndash;5-times higher fructose levels in all areas of the brain compared to controls \u003csup\u003e15\u003c/sup\u003e. Additionally, administration of fructose is associated with increased β-amyloid deposition in animal models of Alzheimer\u0026rsquo;s disease \u003csup\u003e15\u003c/sup\u003e. This indicates that balance of metabolic pathways and energy sources may be important determinants of brain dysfunction.\u003c/p\u003e \u003cp\u003eIn this study, we used unbiased metabolomics to assess the brain of aged mice and the serum of elderly patients following anesthesia/surgery. We examined whether activation of fructose or glucose metabolism in the brain results in increased lactate levels and contributes to onset and progression of dNCR.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eAnimal study\u003c/h2\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003ch2\u003eMice\u003c/h2\u003e\n \u003cp\u003eThis study used 20-month-old C57BL/J6 male mice (Beijing Vital River Laboratory Animal Technology Co. Ltd., Beijing, China). The animal protocol was approved by the Institutional Animal Care and Use Committee at Shanghai Ninth People\u0026apos;s Hospital (SH9H-2022-A937-1), and relevant ARRIVE guidelines were followed.\u003c/p\u003e\n \u003cp\u003eAll mice were first tested in the Y maze to get baseline performance data. Then all mice undergoing surgery, the left carotid artery of mice was exposed under sterile conditions for 30 min under 3% sevoflurane with 40% O\u003csub\u003e2\u003c/sub\u003e at 1 L/min, as done previously \u003csup\u003e16,17\u003c/sup\u003e. Even after the surgery was completed, the mice remained under anesthesia until the duration of total anesthesia lasted for one hour. The anesthetizing chamber was placed in a warm box to maintain mouse rectal temperature at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C. No response to toe pinching was observed during anesthesia. After surgery, all mice received lidocaine ointment. Four days after anesthesia/surgery, mice began training in the Y maze test or were euthanized via decapitation to collect cortical tissues for metabolomics. In total, there are 24 elderly mice. Among them, 10 mice developed dNCR, while 14 mice did not. For metabolomics, we randomly selected 8 cortical tissues samples from a pool of 14 non-dNCR group samples and compared them with 10 cortical tissues samples of dNCR group.\u003c/p\u003e\n \u003cp\u003eTo assess the contribution of metabolic pathways in the mouse model, mice were similarly exposed to anesthesia/surgery as above. However, these experiments also included control mice exposed to only 40% O\u003csub\u003e2\u003c/sub\u003e at a flow of 1 L/min. Mice were injected intraperitoneally with 50 mg/kg fructose metabolism inhibitor 2,5-anhydro-D-mannitol (2,5-AM) thirty minutes before anesthesia/surgery. Samples were collected at 6 hours after completion of anesthesia/surgery to measure lactate and IL-6 levels, and mice were tested the Barnes maze at on the 4th day following anesthesia/surgery, by which time the wound has healed, allowing for the initiation of behavioural testing (n\u0026thinsp;=\u0026thinsp;13; each group).\u003c/p\u003e\n \u003cp\u003eIL-6 knockout mouse experiments were performed in collaboration with Professor Zhongcong Xie at Massachusetts General Hospital. Limited IL-6 knockout mice were available, so experiments were performed with 18-month-old female wildtype (n\u0026thinsp;=\u0026thinsp;4) and IL-6 knockout (n\u0026thinsp;=\u0026thinsp;2) mice. Mice underwent abdominal surgery under 1.5\u0026ndash;2% isoflurane anesthesia. At 6 h after completion of anesthesia/surgery, mice were euthanized to collect their prefrontal cortex.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eIsotope labelling and metabolic flux\u003c/h2\u003e\n \u003cp\u003eAll mice were injected with fructose or glucose intracerebroventricularly. Under anesthesia, the heads of mice were secured in a stereotaxic device, and the epidermis was cut open to locate the Bregma point. From this point, the skull was drilled 0.5 mm towards the tail and 1 mm away from the midline with a grinding drill. An injection needle was inserted at a depth of 2.5 mm to administer 2\u0026ndash;2.5 \u0026micro;L of 2 mg/\u0026micro;L fructose (200 mg/kg; n=) or 0.3 mg/\u0026micro;L glucose (30 mg/kg)\u003csup\u003e18\u003c/sup\u003e. Thirty minutes later, control group mice exposed to only 40% O2 at a flow of 1 L/min for one hour. Anesthesia/surgery group mice undergoing surgery, the left carotid artery of mice was exposed under sterile conditions for 30 min under 3% sevoflurane with 40% O\u003csub\u003e2\u003c/sub\u003e at 1 L/min. Even after the surgery was completed, the mice remained under anesthesia until the duration of total anesthesia lasted for one hour. The incision was sutured, and the ipsilateral cortex was removed to detect metabolic flow after 2 h (due to low downstream metabolite labelling at 1 h). We experimented at multiple time points and found that the optimal time was 2 hours post-surgery.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eWestern blotting\u003c/h2\u003e\n \u003cp\u003eMouse cortical tissues were incubated in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, and inhibitors including sodium pyrophosphate, \u0026beta;-glycerophosphate, EDTA, Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, and leupeptin). Lysates were separated by SDS\u0026ndash;polyacrylamide gel electrophoresis, transferred to PVDF membranes, blocked with 5% non-fat milk, and incubated with primary antibodies against GLUT5 (1:500, 27571-1-AP, Proteintech), MCT1(1:1000, 20139-1-AP, Proteintech), GLUT1 (1:1000, 21829-1-AP, Proteintech), beta-actin (1:20000, 66009-1-Ig, Proteintech), or GAPDH (5174s, Cell Signaling Technology) for 1 h at room temperature. Membranes were visualized with enhanced chemiluminescence substrate (36208ES60, Yeasen Biotechnology, Shanghai, China). Relative target protein levels compared to GAPDH were determined by grey value analysis using Image J.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eBrain lactate and IL-6 levels\u003c/h2\u003e\n \u003cp\u003eMouse brain lactate levels were detected with a lactate kit (SNM184, Biolab Co., Ltd. China) according to manufacturer instructions. Optical density of wells was measured with a fluorescence plate reader (Medical Device, San Jose, CA, USA) at 530 nm. Mouse brain IL-6 levels were detected with an ELISA kit (abs552203, Absin Bioscience, Shanghai China) according to manufacturer instructions. Optical density of wells was measured with a fluorescence plate reader (Medical Device) at 450 nm. Standard curves were generated using R graphing software (4.0.5, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.r-project.org\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eBarnes maze test\u003c/h2\u003e\n \u003cp\u003eMice were tested in the Barnes maze to assess spatial learning and memory \u003csup\u003e19\u003c/sup\u003e. \u003cem\u003ePhase I \u0026ndash; Habituation\u003c/em\u003e: Mice were habituated to the environment for at least 30 min. Light intensity was set to ~\u0026thinsp;1000 Lux. Speakers were activated, and aversive noise was set to 90 dB. Mice were situated in the centre of a circular platform with 20 evenly spaced holes. Mice were guided with a black lid to the escape hole and remained there for 2 min. Mice rested for at least 30 min. The same process was repeated daily. \u003cem\u003ePhase II - Training\u003c/em\u003e: Mice were placed in the centre and covered with a black lid for 15 s. The black lid was removed while turning on the aversive noise. The experiment lasted 3 min. If the mouse successfully entered the escape hole, the aversive noise stopped and the experiment ended. The mouse was allowed to stay in the escape tunnel for 15 s before removal. If the mouse failed to find the escape hole, it was guided there with a black lid and allowed to stay there for 15 s. Consistent with a previous study \u003csup\u003e16\u003c/sup\u003e, mice began training 4 days after anesthesia/surgery, and spatial memory tests were conducted 5 days after training. The EthoVision-XT video tracking system (Noldus, Wageningen, and the Netherlands) was used to monitor mice in the maze. dNCR was defined as postoperative Y maze scores\u0026thinsp;\u0026gt;\u0026thinsp;5% decreased compared to preoperative scores.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eClinical study\u003c/h2\u003e\n \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\n \u003ch2\u003eStudy enrolment\u003c/h2\u003e\n \u003cp\u003eThis prospective observational cohort study was conducted at Shanghai Tongji Hospital at Tongji University in 2021\u0026ndash;2022. After receiving approval from the ethics committee (2021\u0026thinsp;\u0026minus;\u0026thinsp;111) as well as written informed consent, we recruited 43 patients. Preoperative inclusion criteria were: 1)\u0026thinsp;\u0026ge;\u0026thinsp;60 years old; 2) undergoing optional spinal surgery; 3) American Society of Anesthesiologists physical status of I\u0026ndash;III; and 4) Mini-Mental State Examination score higher than the minimum score for the patient\u0026rsquo;s education level [\u0026ge;\u0026thinsp;20 for primary school (\u0026le;\u0026thinsp;6 years of education), or \u0026ge;\u0026thinsp;23 for middle school and higher (\u0026gt;\u0026thinsp;6 years of education)]. Preoperative exclusion criteria were: 1) hearing, vision, or speech barriers that prevented cooperation with perioperative neuropsychological tests; 2) nervous system disease or mental illness; 3) serious systemic diseases or severe postoperative complications (e.g., bleeding, cerebral infarction, pulmonary infection); or 4) metal implants, artificial cochlear implants, cardiac pacemakers, and other implanted devices. This study adhered to STROBE guidelines. The study protocol was registered with chictr.org.cn (ChiCTR2200057080).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eAnesthesia, surgery, and serum sample collection\u003c/h2\u003e\n \u003cp\u003eAll patients received general anesthesia under tracheal intubation. The induction drugs used were sufentanil (20\u0026ndash;30 \u0026micro;g), etomidate (0.3\u0026ndash;0.4 mg/kg), and cis-atracurium (0.2 mg/kg). A preoperative antiemetic (e.g., ramosetron) was used as required. Intraoperative intravenous inhalation anesthesia used was sevoflurane (1\u0026ndash;1.5%) combined with propofol (2\u0026ndash;6 mg/kg/h) and remifentanil (0.05\u0026ndash;1 \u0026micro;g/kg/min). Sufentanil and cis-atracurium were used intermittently to deepen anesthesia and relax muscles as needed. Patient anesthesia control was according to bleeding volume, urine volume, and arterial blood pressure. Timely replenishment of crystalloid (lactated Ringer\u0026apos;s) and colloid (succinyl gelatin) intravenous solutions was maintained to monitor blood cell production. We used a Fabius GS premium anesthetic workstation (Dr\u0026auml;ger, L\u0026uuml;beck, Germany) for general anesthesia, a multifunctional monitor (GE Carescape B850, Boston, MA, USA) to measure the patient\u0026rsquo;s vital signs, and anesthesia depth monitor (ConView YY-106, Pearlcare, Zhejiang, China) to determine depth of anesthesia. Intraoperative blood gas analysis (GEM Premier 3500, Werfen, Spain) was used to monitor blood oxygen and adjust water\u0026ndash;electrolyte balance. All participants received standardized perioperative care, including postoperative pain management using sufentanil (100 \u0026micro;g/48 h)/hydromorphone (8 mg/48 h) patient-controlled analgesia.\u003c/p\u003e\n \u003cp\u003eWe collected 5 mL of venous blood from the internal jugular vein of participants before and immediately after surgery. Separation gel and coagulant were added to blood tubes (20231280, Shanghai Orsin Medical Technology Co. Ltd., Shanghai, China). Samples were incubated at 25\u0026deg;C for 30 min and centrifuged at 3000 rpm for 15 min at 4\u0026deg;C. Serum supernatant was collected and stored at -80\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003edNCR measurement\u003c/h2\u003e\n \u003cp\u003eTrained clinical researchers assessed participants for cognition change before surgery and prior to being discharged or at 7 days post-surgery using the Digit Symbol Substitution Test, Trail Making Test A, Judgment of Line Orientation Test, Stroop Color Word Test, and Auditory Verbal Learning Test, with a 3 min gap between intervals. At 30 days post-surgery, the Abbreviated Mental Test Score was assessed over the phone. A decrease of one SD in both evaluation scales after surgery was considered dNCR \u003csup\u003e20\u003c/sup\u003e. Patients who did not develop dNCR constituted the control group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eLiquid chromatography\u0026ndash;tandem mass spectrometry\u003c/h2\u003e\n \u003cp\u003eConsistent with a previous study \u003csup\u003e21\u003c/sup\u003e, 50 \u0026micro;L of patient serum and 300 \u0026micro;L of methanol were mixed by vortexing for 10 min at 4\u0026deg;C and 2000 rpm. After centrifugation at 4\u0026deg;C for 10 min at 12,000 rpm, 300 \u0026micro;L of supernatant were transferred to a new tube and dried using a stream of nitrogen. Dry residue was reconstituted in 50 \u0026micro;L of 2% acetonitrile, mixed by vortexing at 4\u0026deg;C for 10 min at 2000 rpm, and centrifuged at 4\u0026deg;C for 10 min at 12,000 rpm. The supernatant was transferred to an autosampler vial. Sample extracts were analysed by reversed-phase chromatography using a mobile phase of water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid) at a constant rate of 0.20 mL/min. The injection volume was 1 \u0026micro;L, and the temperature of the autosampler was 40\u0026deg;С. Chromatographic separation was achieved on an Acquity UPLC HSS T3 column (2.1 \u0026times; 100 mm, 1.8 \u0026micro;m, Waters Corp., Milford, MA, USA) on a Vanquish system (Thermo Fisher Scientific, Waltham, MA, USA). A TSQ Altis mass spectrometer (Thermo Fisher Scientific) with electrospray ionization source was operated in multiple reaction monitoring mode for mass data acquisition. The parameters of the heated electrospray ionization source were: sheath gas at 40 arbitrary units, aux gas at 10 arbitrary units, spray voltage at 3.5 kV (+)/2.5 kV (-), ion transfer tube temperature at 320\u0026deg;C, and vaporizer temperature at 325\u0026deg;C. Optimized mass spectrometer conditions were used for quantitative analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003eClinical data\u003c/h2\u003e\n \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD if normally distributed; otherwise, they are expressed as median and 25th and 75th percentiles. Differences between groups were analysed using student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, Wilcoxon test, or Fisher\u0026apos;s exact test as appropriate. Mean and SD of patients\u0026rsquo; preoperative and postoperative scale scores were calculated.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eMetabolomics data\u003c/h2\u003e\n \u003cp\u003eWidely targeted metabolomics data included 543 metabolites from 100 samples. The online analytical tool MetaboAnalyst 5.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.metaboanalyst.ca/\u003c/span\u003e\u003c/span\u003e) was used to perform multivariate (multidimensional) statistical analysis. Multivariate statistical methods such as principal component analysis, partial least squares discrimination analysis, and orthogonal partial least squares discrimination analysis were used for comparative analysis of the whole metabolic spectrum and metabolite screening. Data were normalized before multivariate statistical modelling. Differential metabolites were selected using criteria of variable importance in projection (VIP)\u0026thinsp;\u0026gt;\u0026thinsp;1.0 and student\u0026apos;s t-test \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBaseline participant characteristics\u003c/h2\u003e \u003cp\u003eThe only baseline metrics that significantly differed between dNCR and non-dNCR groups were blood glucose (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.046) and blood creatinine (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.052) (Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003edNCR patients have significantly decreased postoperative serum fructose 1-phosphate levels\u003c/h2\u003e \u003cp\u003eAmong 43 TJ participants, 17 patients developed dNCR. We applied orthogonal partial least squares discrimination analysis to postoperative serum metabolomics data for 543 metabolites between dNCR and non-dNCR groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Based on VIP values, we ranked the top differential metabolites between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Postoperative serum levels of fructose 1-phosphate\u0026mdash;which can be directly converted into lactate\u0026mdash;were significantly decreased in dNCR compared to non-dNCR groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC,D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003edNCR mice have significantly decreased postoperative brain fructose 1-phosphate levels\u003c/h2\u003e \u003cp\u003eWe investigated whether fructose 1-phosphate levels also were associated with dNCR in our mouse model. At four days post-anesthesia/surgery, mice were tested in the Y maze to assess postoperative cognitive impairment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Among 24 mice, 10 developed dNCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We applied orthogonal partial least squares discrimination analysis to postoperative brain metabolomics data for 543 metabolites in the brain of mice in dNCR (n\u0026thinsp;=\u0026thinsp;10) and non-dNCR groups (n\u0026thinsp;=\u0026thinsp;8, randomly chosen) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). We identified differential metabolites between dNCR and non-dNCR groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), including significantly decreased fructose 1-phosphate levels in dNCR compared to non-dNCR mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE,F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDecreased metabolites can be attributed to either metabolic decreased yield or increased consumption. A member of the facilitative glucose transporter (GLUT) family, GLUT5, is responsible for passive membrane transport of fructose \u003csup\u003e22\u003c/sup\u003e. GLUT5 is the only transporter specific for fructose and cannot transport glucose or galactose \u003csup\u003e23\u003c/sup\u003e. Interestingly, GLUT5 levels were increased in dNCR compared to non-dNCR groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG,H).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eElevated lactate levels are associated with activation of fructose metabolism\u003c/h2\u003e \u003cp\u003eWe investigated whether the increased lactate levels in mice were due to fructose metabolism or glucose metabolism. Fructose can enter glycolysis through two pathways: the fructokinase pathway, or the hexokinase pathway \u003csup\u003e24\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). At 2 h after anesthesia/surgery, mouse brains had increased levels of downstream fructose decomposition products (e.g., fructose 6-phosphate, fructose 1-phosphate) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB,C) as well as increased lactate production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Glucose also can enter glycolysis directly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), although altered glucose to lactate metabolism was not observed following anesthesia/surgery compared to control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u0026ndash;H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then used intraperitoneal injection of fructose metabolism inhibitor 2,5-anhydro-D-mannitol (2,5-AM) \u003csup\u003e25\u003c/sup\u003e to determine the effect of fructose metabolism on anesthesia/surgery-induced learning and memory dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Administration of 2,5-AM prevented the increased lactate levels in the brains of elderly mice following anesthesia/surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). While mice exposed to anesthesia/surgery had longer latency to find the target and spent less time in the entry zone of the Barnes maze compared to control mice, administration of 2,5-AM also prevented these deficits (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnesthesia/surgery-induced elevation of IL-6 may be related to fructose metabolism in the brains of elderly mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA high-fructose diet increases neuroinflammation \u003csup\u003e14\u003c/sup\u003e, and neuroinflammation contributes to cognitive impairment in rodents following surgery under general anesthesia \u003csup\u003e26\u003c/sup\u003e. In addition, inflammation also activates fructose metabolism and especially IL-6 \u003csup\u003e27\u0026ndash;29\u003c/sup\u003e. IL-6 is both necessary and sufficient to produce cognitive decline in mice \u003csup\u003e26,30\u003c/sup\u003e and patients \u003csup\u003e1\u003c/sup\u003e. To clarify the upstream and downstream connections between neuroinflammation and fructose metabolism, we injected the fructose metabolism inhibitor 2,5-AM into the abdominal cavity of elderly mice before anesthesia/surgery. Previous research shows that abdominal surgery can lead to postoperative cognitive impairment in mice\u003csup\u003e31\u003c/sup\u003e. While anesthesia/surgery significantly increased IL-6 levels in the brains of mice, fructose metabolism inhibitors only had a slight salvage effect on elevated IL-6 levels following anesthesia/surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, IL-6 knockout transgenic mice exhibited decreased expression of \u003cem\u003eGlut5\u003c/em\u003e in the brain after anesthesia/surgery compared to wildtype mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Surprisingly, expression of glucose transmembrane transport protein GLUT1, which serves as a crucial gating protein to control passage of glucose into and out of tissues, was unaffected by IL-6 knockout. In addition, lactate exchange is facilitated by MCT1, which handles uptake of exogenous lactate by glycolysis \u003csup\u003e32\u003c/sup\u003e. IL-6 knockout transgenic mice exhibited decreased \u003cem\u003eMct1\u003c/em\u003e expression in the brain after anesthesia/surgery compared to wildtype mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study shows that elderly patients who underwent anesthesia/surgery and developed dNCR had decreased fructose 1-phosphate levels in their blood compared to those who did not develop dNCR. We found similar results in our mouse model. Further, mice in the dNCR group had increased \u003cem\u003eGlut5\u003c/em\u003e expression compared to the non-dNCR group, suggesting anesthesia/surgery activates fructose metabolism. Metabolic flux experiments showed that activation of fructose metabolism increased glycolysis and lactate production, and that fructose inhibitors could inhibit the anesthesia/surgery-induced increase in lactate. Further, administering fructose inhibitors to mice helped prevent the postoperative cognitive decline induced by anesthesia/surgery. In addition, our results indicate that IL-6 could be the upstream mechanism to activate fructose metabolism. Taken together, these data suggest that anesthesia/surgery activates fructose metabolism in the brain to increase lactate levels, contributing to dNCR.\u003c/p\u003e \u003cp\u003eFructose can be converted into fructose 6-phosphate by hexokinase and then enter glycolysis. Phosphate fructose kinase serves as the rate-limiting enzyme in this step. However, fructose also can be converted by fructose kinase (ketohexokinase) into fructose 1-phosphate, which also participates in glycolysis. Notably, there is no rate-limiting enzyme involved in this pathway. Fructose is more readily converted to lactate than glucose. We determined that anesthesia/surgery triggered fructose metabolism and increased fructose 1-phosphate levels, causing fructose to enter glycolysis and produce lactate. Fructose 1-phosphate and fructose metabolism thus may serve as potential therapeutic targets for dNCR.\u003c/p\u003e \u003cp\u003eFructose activation is potentially neurotoxic through various mechanisms. First, conversion of fructose into fat is a simple process \u003csup\u003e33\u003c/sup\u003e. The liver plays a crucial role in metabolizing fructose, but excessive consumption can result in low high-density lipoprotein cholesterol levels and high hepatic inflammation \u003csup\u003e34\u003c/sup\u003e. In addition, dysfunctional lipid metabolism in astrocytes is crucial in dNCR development \u003csup\u003e35\u003c/sup\u003e. Second, high fructose consumption disrupts brain mitochondrial function and may contribute to neurological disorders. In both short-term \u003csup\u003e36\u003c/sup\u003e and long-term \u003csup\u003e37\u003c/sup\u003e experiments, high fructose consumption results in significantly decreased levels of major mitochondrial functional markers in the brain. High fructose consumption changes the brain\u0026rsquo;s unfolded protein response and macroautophagic machinery, promoting build-up of aggregates of β-amyloid 1\u0026ndash;42 (Aβ42), tau-p-S199, and tau-p-S404, which are indicators of neurodegeneration \u003csup\u003e38\u003c/sup\u003e. Third, activation of fructose metabolism can result in insulin resistance \u003csup\u003e12\u003c/sup\u003e. Clinical postoperative cognitive dysfunction can be effectively predicted by preoperative insulin resistance \u003csup\u003e39,40\u003c/sup\u003e or postoperative insulin resistance\u003csup\u003e3\u003c/sup\u003e indicating that targeted prevention and treatment strategies for insulin resistance may be effective interventions for at-risk patient\u003c/p\u003e \u003cp\u003eDespite our significant findings, this study is limited in that our experimental design assessed mice at 4 days after anesthesia/surgery, but the increase in fructose 1-phosphate may have occurred earlier. A previous clinical study shows that fructose levels surge in cerebrospinal fluid at 2 days post-anesthesia/surgery \u003csup\u003e8\u003c/sup\u003e. Yet that study was limited by its sample size, so perioperative cognitive tests were not performed and the association between fructose metabolism and perioperative neurocognitive disorder was not clear. Yet we believe that study and our study are complementary, showing correlation between fructose metabolism activated by anesthesia/surgery and postoperative dNCR. In addition, we used IL-6 knockout transgenic mice to indicate that the increased IL-6 caused by anesthesia/surgery may trigger fructose metabolism. Unfortunately, we only had two 18-month-old female elderly IL-6 knockout transgenic mice, so they cannot be statistically analysed. Nevertheless, given that elderly IL-6 knockout transgenic mice exhibited significantly decreased \u003cem\u003eGlut5\u003c/em\u003e expression in the brain after anesthesia/surgery, it is feasible that neuroinflammation acts as an upstream mechanism to activate fructose metabolism. That study was limited by it did not pinpoint the origin of fructose. Typically derived from food, fructose is primarily stored in the intestines and liver. Moreover, clinical patients often undergo fasting and hydration before anesthesia. It has been discovered that the brain generates endogenous fructose, leading to speculation that anesthesia and surgery may trigger the conversion of other substances like glutamine into fructose, thus impacting fructose metabolism. Future research may focus on uncovering the source of fructose.\u003c/p\u003e \u003cp\u003eIn conclusion, our results indicate that anesthesia/surgery-induced neuroinflammation can reprogram glucose metabolism, ultimately leading to dNCR. This reprogramming may be driven by increased IL-6 levels in the brain to activate fructose metabolism. Downstream metabolites of fructose then enter glycolysis, producing excessive lactate and ultimately contributing to postoperative cognitive impairment. Thus, fructose metabolism may be a potential treatment for postoperative neurocognitive disorder.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e This research was supported by the National Natural Science Foundation of China (82171173) and Clinical Research Program of 9\u003csup\u003eth\u003c/sup\u003e People\u0026apos;s Hospital affiliated with Shanghai Jiao Tong University School of Medicine (JYLJ202221, JYLJ202304).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests:\u003c/strong\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003eThe study protocol was registered with chictr.org.cn (ChiCTR2200057080)\u003c/p\u003e\n\u003cp\u003ehttps://www.chictr.org.cn/showproj.html?proj=152662\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJustification of authorship\u003c/strong\u003e: LZ:\u0026nbsp;conceptualization, investigation, writing \u0026ndash; reviewing and editing, funding acquisition. JH L, JH L, JY Z: patient recruitment, data collection. RZ: software, validation. ZJ M, HW, and XL: methodology. JY: data curation. HJ: conceptualization, writing \u0026ndash; reviewing and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during this study are not publicly available but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e This research was supported by the National Natural Science Foundation of China (82171173) and Clinical Research Program of 9\u003csup\u003eth\u003c/sup\u003e People\u0026apos;s Hospital affiliated with Shanghai Jiao Tong University School of Medicine (JYLJ202221, JYLJ202304).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLi Y, Chen D, Wang H, et al. 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High-Fructose Consumption Impairs the Redox System and Protein Quality Control in the Brain of Syrian Hamsters: Therapeutic Effects of Melatonin. \u003cem\u003eMol Neurobiol\u003c/em\u003e. Oct 2018;55(10):7973-7986. doi:10.1007/s12035-018-0967-2\u003c/li\u003e\n \u003cli\u003eHe X, Long G, Quan C, Zhang B, Chen J, Ouyang W. Insulin Resistance Predicts Postoperative Cognitive Dysfunction in Elderly Gastrointestinal Patients. \u003cem\u003eFront Aging Neurosci\u003c/em\u003e. 2019;11:197. doi:10.3389/fnagi.2019.00197\u003c/li\u003e\n \u003cli\u003eTang N, Jiang R, Wang X, et al. Insulin resistance plays a potential role in postoperative cognitive dysfunction in patients following cardiac valve surgery. \u003cem\u003eBrain Res\u003c/em\u003e. Feb 15 2017;1657:377-382. doi:10.1016/j.brainres.2016.12.027\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"862faed1-f739-4032-915d-29e12483d874","identifier":"10.13039/501100001809","name":"National Natural Science Foundation of China","awardNumber":"82171173","order_by":0},{"identity":"ff2d1fbd-1d0d-42aa-a4ac-c9afd7336a8e","identifier":"10.13039/501100004921","name":"Shanghai Jiao Tong University","awardNumber":"JYLJ202221","order_by":1},{"identity":"97f0f0d3-4680-4e4c-8d32-aa099f84720c","identifier":"10.13039/501100004921","name":"Shanghai Jiao Tong University","awardNumber":"JYLJ202304","order_by":2}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Shanghai Jiao Tong University","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":"general anesthesia, delayed neurocognitive recovery, fructose, metabolomics, lactate","lastPublishedDoi":"10.21203/rs.3.rs-4724665/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4724665/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Elderly individuals display excessive lactate levels that may contribute to development of cognitive impairment following surgery, including delayed neurocognitive recovery (dNCR). Since the origin of this increased lactate is unknown, here we assessed associations between metabolic pathways and postoperative dNCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e This study included 43 patients (≥65 years old) who had surgery under general anaesthesia. We also used a mouse model in which 20-month-old mice were exposed under sevoflurane to induce postoperative dNCR. Metabolomics were used to measure metabolites in the serum of patients and brains of mice following anaesthesia/surgery. Isotope labelling and metabolic flux were used to analyse flow and distribution of specific metabolites in metabolic pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Among 43 patients, 17 developed dNCR. Metabolomics showed significantly decreased postoperative serum fructose 1-phosphate levels in dNCR compared to non-dNCR patients. Similar results were found in the mouse model. Isotope labelling and metabolic flux experiments in mice showed fructose but not glucose entered glycolysis, increasing lactate levels after anaesthesia/surgery. Administration of intraperitoneal fructose inhibitors to mice effectively inhibited the increased lactate levels and cognitive dysfunction following anaesthesia/surgery. We also found anaesthesia/surgery increased IL-6 levels in mice, and that IL-6 may function upstream in fructose activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e These results suggest that anaesthesia/surgery activates fructose metabolism, producing excessive lactate and ultimately contributing to postoperative cognitive impairment. Fructose metabolism is thus a potential therapeutic target for dNCR.\u003c/p\u003e","manuscriptTitle":"Fructose metabolism is associated with anesthesia/surgery induced lactate production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-15 18:45:33","doi":"10.21203/rs.3.rs-4724665/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":"fb10988a-d7e3-42b8-b6ac-fd2918bff5a4","owner":[],"postedDate":"July 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34460749,"name":"Anesthesiology \u0026 Pain Medicine"}],"tags":[],"updatedAt":"2024-07-15T18:45:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-15 18:45:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4724665","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4724665","identity":"rs-4724665","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}