Pain hypersensitivity and increased urinary tetrahydrobiopterin levels in mice submitted to high fat diet

Pain hypersensitivity and increased urinary tetrahydrobiopterin levels in mice submitted to high fat diet | 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 Pain hypersensitivity and increased urinary tetrahydrobiopterin levels in mice submitted to high fat diet Tuany Eichwald, Leonardo Barros, Alexandre Francisco Solano, Débora da Luz Scheffer, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4458806/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 Objective: This study aimed to examine the impact of high-fat diet (HFD)-induced obesity on pain sensitivity and tetrahydrobiopterin (BH4) levels. The effect of moderate-intensity physical exercise, an anti-inflammatory non-pharmacological intervention, on pain scores was also investigated. Methods: Adult male C57BL/J6 mice were fed standard or HFD for eight weeks. Total body weight, food intake, locomotor and motivational behavior and pain reflexes were measured. A subgroup of animals underwent physical exercise for five days per week over six weeks. Blood was collected for glucose tolerance testing and levels of lactate. Urine samples were collected to measure BH4 levels. Results: We showed that HFD increased weight gain, visceral white adipose tissue, and the percentage of body fat. These anthropometric alterations were characterized by impaired glucose tolerance at four and eight weeks of the dietary intervention. It was also observed reduced locomotor activity and higher pain scores in HFD-fed mice that were prevented by the physical exercise intervention. HFD also induced the increase of urinary BH4 levels at four and eight weeks of intervention. Conclusion: Urinary BH4 can be proposed as a potential easy-to-access, sensitive and reliable biomarker of pain development, and a promising target for the control of pain hypersensitivity in obesity. obesity adipose tissue sepiapterin reductase inhibitors chronic pain biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Obesity is defined by the World Health Organization (WHO) as abnormal and excessive accumulation of fat (WHO 2021 ). The increase in white adipose tissue (WAT) from a daily positive energy balance is associated with low-grade chronic systemic inflammation, with immune infiltration in the WAT. Obesity is a multifactorial etiology disease, and the clinical manifestations involve chronic pain and joint dysfunction (Thomazeau et al. 2014 ). Obesity predisposes the development of a large number of chronic diseases, including inflammatory and degenerative conditions of the musculoskeletal system and the peripheral and central nervous systems (Cope et al. 2018; Smith et al. 2011 ). Indeed, the inflammatory pathways are persistently activated in several brain regions controlling peripheral regulation of energy, glucose, and lipid metabolism (Gaspar et al. 2010 ; Hotamisligil 2017 ). Tetrahydrobiopterin (BH4) is a pterin that acts biologically as an mandatory cofactor for the metabolism of phenylalanine and certain lipids, and for the biosynthesis of the neurotransmitters dopamine, serotonin and nitric oxide (NO) (Werner et al. 1990 ; Werner, Blau, and Thöny 2013 ). BH4 intracellular concentrations are finely tuned by three metabolic pathways, assuring continue basal levels of the molecule to supports the systems where it is involved (see Eichwald et al. 2023 for a review). However, excessively increased BH4 levels have been associated with numerous pathological conditions, including cardiovascular disease (Bendall et al. 2014 ), cancer (Cronin et al. 2018 ), and chronic pain (Latremoliere et al. 2015 ; Tegeder et al. 2006 ). Furthermore, inflammatory conditions positively modulate the synthesis of BH4, in immune, nerve cells, and others (Ghisoni et al. 2015 ; de Paula Martins et al. 2018 ). Pain is a conscious experience that demands cortical participation and aversive information from nociception, processed by the peripheral nervous system, and unconsciously modulated by the central nervous system (Woolf and Ma 2007). Normally, the adaptive response against the stimuli that activate both systems overcome the threat and reach the resolution. However, maladaptive inflammatory reactions, in which pro-inflammatory mediators persistently activate and sensitize neurons at different levels of the nociceptive pathway, are believed to induce chronic pain (Costigan et al. 2009 ; Eichwald and Talbot 2020). Thus, this work aimed to identify whether obesity, a low-degree chronic inflammatory disease, may negatively modulate nociceptive thresholds and positively increase BH4 levels that can be monitored in biological fluids as a biomarker for pain. 2 Material and methods 2.1 Animals Adult male C57BL/J6 mice (3–5 months of age; 45–50 g) from the Centre for Biological Sciences, Universidade Federal de Santa Catarina (UFSC) (Brazil) were acclimated for ten days in a controlled environment (22 ± 1°C, 12 h light/dark cycle) with free access to water and food. All experimental protocols, approved by UFSC’s Ethics Committee for Animal Research (CEUA, 4401201118), complied with current guidelines for laboratory animals and ethical care and ethical standards for experimental pain research in conscious animals (Percie du Sert et al. 2020). 2.2 Experimental strategy Mice were randomly divided into two groups. One group received a high-fat diet (HFD), while the other group received a standard diet. Both groups were maintained on their respective diets for 8 weeks. Mice were fed with HFD (n = 26) to induce increased adiposity, and to be used as a proxy of human obesity. The diet’s macronutrient composition was proteins: 20 kcal %; carbohydrates: 35 kcal %; lipids: 45 kcal % (Pragsoluções Biociências, Jaú, São Paulo, Brazil). Animals fed with standard diet were used as controls (n = 12). The standard diet composition was protein: 20 kcal %; carbohydrates: 70 kcal %; lipids: 10 kcal %. The animals were housed in cages containing 4–5 individuals from the same experimental group. Cages were labeled with the type of treatment (diets), and the corresponding food was provided. During the whole experimental strategy, mice were closely monitored for humane endpoints. Experimental groups consisted of n = 6–7 mice. This number allowed to perform the behavioral tests with sensitivity and reproducibility and to collect tissues generating an appropriate unit of mass to perform the biochemical measurements. This number of sample size was calculated by applying “power statistics”, as shown below. Sample size calculation : We used the following formula for sample size calculation for the comparison between two groups with quantitative data endpoints: Sample size = 2 SD 2 (Z a/2 + Z β ) 2 /d 2 . Where: Standard deviation (SD) = estimated from previous studies Z a/2 = Z 0.05/2 = Z 0.025 = 1.96 (From Z table) at type 1 error of 5% Z β = Z 0.20 = 0.842 (From Z table) at 80% power d = effect size = difference between mean values Hence now formula will be: Sample size = 2 SD 2 (1.96 + 0.842) 2 /d 2 Based on previous studies from our group we have the following assumptions: - The minimum difference between groups’ mean will be set at least at 54.4%; - Biological experiments inherently have 10–15% error margin; - Differences less than 20% of each group’s mean can increase the probability of type I or type II errors; - The standard deviation is typically 35% of the mean value. Sample size = 2 35 2 (1.96 + 0.842) 2 /54.4 2 = 6.5. 2.3 Intraperitoneal glucose tolerance test The glucose tolerance test (GTT) was performed after 6 h of fasting by injecting mice with 2 g/kg glucose intraperitoneally (i.p.). Blood glucose levels were measured at 0, 5, 10, 15, 30, 60 and 120 min from tail blood samples (adapted from Rafacho et al. 2008 ). The test was performed before the dietary intervention, at 4 and 8 weeks. 2.4 Physical exercise To prevent HFD-induced WAT accumulation, a group of animals was also submitted to physical exercise as described below. 2.4.1 Incremental test to determine the maximal capacity for exercise The incremental test was performed to identify 60% of the maximal capacity of the animals ( Supplementary Fig. 1a ) (Aguiar et al. 2016 ). When exhaustion was reached, caudal blood was collected to measure lactate levels. During the first week of training, performance was scored daily: 1 for animals that refused to run; 2 for animals that ran at variable speeds, run, and stop; 3 for animals that ran regularly; 4 for animals that are runners; 5 for animals that are good runners. Only animals that scored 3 or more continued the training. Two animals scored less than 3 and were discontinued from the physical exercise protocol. 2.4.2 Physical exercise protocol The physical exercise protocol consisted of five training sessions/week for six weeks on a treadmill with an interval of 48 h each week (Table 1 ) (Aguiar et al. 2016 ). Animals began with a 5 min warm-up at 40% intensity before each session. Training speed was set at 60% of the final speed from the incremental test, indicating moderate intensity. Sessions lasted 35, 40, and 45 min/day for the first 3 weeks, with a subsequent incremental test. The last 2 weeks sessions lasted 35 and 40 min/day. Table 1 Physical exercise protocol Training weeks Session duration (min) Treadmill inclination (%) Activity intensity (%) 1 35 2 60 2 40 2 60 3 45 2 60 Effort test to equalize intensities 5 35 2 60 6 40 2 60 2.4.2.1 Caudal blood collection Immediately at the end of the incremental test, caudal blood was collected in tubes containing sodium fluoride to inhibit the glycolysis (Rhoden and Rhoden 2006). Blood was used to measure lactate concentrations. 2.4.2.2 Lactate measurement The specific analyzer YSL 2700 (YSL 2700, Yellow Springs, CA, USA) was used to quantify blood lactate levels. High blood lactate indicated exercise protocol’s intensity. 2.5 Behavioral tests Animals were acclimated for 1 h before behavioral tasks in the experimental room. Assessments occurred during the rodent's light phase and were conducted by a researcher blinded to the experimental groups. 2.5.1 Locomotor activity Locomotor activity was evaluated in a 100 cm×100 cm×50 cm open field arena in a sound-attenuated room under low-intensity light. Each animal’s exploratory activity was video recorded for 5 min, and analyzed using the ANY-mazy Platform™ (Aguiar et al. 2013 ). Animals were evaluated before and after the dietary intervention. 2.5.2 Motivational behavior Motivational behavior was assessed by measuring for 5 min the grooming behavior after mice being squirted with 1 mL of a 10% sucrose solution on their dorsal coat (Scheffer et al. 2021 ). Animals were assessed before and after the dietary intervention. 2.5.3 Mechanical hypersensitivity To assess mechanical sensitivity, the withdrawal threshold was measured using a series of von Frey filaments (0.20, 0.40, 0.70, 1.6, 3.9, 5.9, 9.8 and 13.7 mN, Stoelting, Wood Dale, IL, USA; equivalent in grams to 0.02, 0.04, 0.07, 0.16, 0.40, 0.60, 1.0 and 1.4). The 50% withdrawal threshold was determined using the ‘up-down’ method and calculated using Up-Down Reader software (Gonzalez-Cano et al. 2018 ). Animals were evaluated before and 2, 4, 6, and 8 weeks after the dietary intervention. 2.5.4 Thermal hypersensitivity Mice were placed in an acrylic cylinder on the surface of a previously heated metal plate (50 ± 2°C) (INSIGHT®). The latency that the animal took to stand up, shake and/or lick one of the hind legs was considered as an indication of thermal hypersensitivity. Animals were evaluated after 8 weeks of dietary intervention. 2.5.5 Chemical hypersensitivity Animals received 20 uL injection containing 1.6 µg capsaicin under the skin of the dorsal right hind paw. The animal was then placed in a transparent glass chamber and observed for 5 min. The latency and the number of times of licking and/or shaking the paw, fingers, or leg where the capsaicin was injected was timed (Sakurada et al. 1992 ). Animals were assessed after the dietary intervention. 2.6 Adipose tissue dissection Animals were euthanized at the end of 8 weeks of dietary intervention, following the ARRIVE guidelines, to collect blood and visceral WAT (vWAT). 2.7 BH4 quantification Urinary BH4 levels were determined by high-performance liquid chromatography (HPLC) (Alliance e2695, Waters, MA, USA) coupled with electrochemical detection as previously described with some modifications (Latremoliere et al. 2015 ). The results were expressed as µmol/mmol of creatinine. 2.7.1 Quantification of creatinine Urinary creatinine concentrations were determined using a commercial kit (Pointe Scientific Inc., Canton, Michigan, USA). Creatinine levels were indicated as mmol/L. 2.8 Statistical analysis Data are presented as mean ± standard error of mean. Data were analyzed by one-way or two-way ANOVA followed by the post hoc test of Šídák when F was significant. When comparing two independent groups, one tail Student’s t test was used. The accepted level of significance for the tests was P ≤ 0.05. Statistics and all graphs were performed by using GraphPad Prism 9®. 3 Results Figure 1 shows the effect of HFD on caloric consumption, body weight and fat accumulation. Figure 1a shows that caloric consumption was significantly lower in animals submitted to HFD. However, Figure 1b shows that the weight gain delta, calculated as the final body weight minus the initial body weight, was increased in HFD fed mice [ t (11) = 1.77; P ≤ 0.05]. In agreement, the energy efficiency of the diets ( Figure 1c ) was shown to be higher in HFD-fed mice [ t (11) = 2.28; P < 0.05]. Figure 1d shows that the weight of the vWAT was significantly higher in HFD-fed animals [ t (11) = 3.38; P < 0.01]. Similarly, the relationship between vWAT and total body weight was increased in the group of animals fed with HFD ( Figure 1e ) [ t (11) = 4.42; P < 0.001]. Supplementary Figures 2a and 2b are representative images from a mouse from each experimental group. GTT and the fasting glucose test were performed at three different times, baseline (pre-intervention), 4 and 8 weeks after intervention to identify insulin resistance (Fig. 2 ). Figure 2 a shows that GTT was identical in both experimental groups before the dietary intervention. The area under the curve (Fig. 2 b) and fasting glucose levels (Fig. 2 c) were also not different. Figure 2 d shows that GTT was not altered after 4 weeks of intervention; however, the area under the curve shown in Fig. 2 e was significantly bigger in the HFD group [ t (11) = 4.90; P < 0.001], as well as fasting blood glucose levels (Fig. 2 f) [ t (11) = 1.903; P ≤ 0.05]. Figure 2 g shows significant increase in glycemia at 15 min [F (6,66) = 4.68; P < 0.001], and the values under the curve [ t (11) = 2.774; P < 0.01] (Fig. 2 h) after the glucose injection in HFD-fed mice. However, fasting glycemia at week 8 (Fig. 2 i) was not different between groups. Figure 3 shows the influence of HFD on motivational behavior and spontaneous locomotor activity. Figures 3 a-c show that the performance in the sucrose test, used to assess motivation, depression, and anhedonia (Fig. 3 a), and total time spent in grooming (Fig. 3 c) were not altered in HFD-fed mice. However, grooming total time spent was longer in the group that received the standard diet after 8 weeks of intervention [ F (2,20) = 0.80; P ≤ 0.05] (Fig. 3 b). To identify whether the diet would cause changes in locomotor activity, the open field test was performed before the intervention and at the end of 8 weeks of experimentation. Figure 3 h shows that animals fed with HFD for 8 weeks spent less time in the center of the apparatus [ F (2,23) = 0.11; P ≤ 0.05]. No significant differences were observed between groups in total distance traveled (Fig. 3 d), crossing (Fig. 3 e), average speed (Fig. 3 f), maximal speed (Fig. 3 g), and time in the periphery (Fig. 3 i). To investigate whether the HFD would induce nociceptive changes under different noxious stimuli, mice were evaluated for chemical, thermal and mechanical hyperalgesia. Figure 4 a shows that there was no difference between groups in the latency for the animals' first response evoked by capsaicin. However, total response time evoked by capsaicin was higher in animals fed with HFD [ t (9) = 2.11; P ≤ 0.05] (Fig. 4 b). Figure 4 c shows that there was no significant difference in the reflexes evoked by the thermal stimulus. HFD-fed mice showed lower thresholds for mechanical hyperalgesia at weeks 2, 6 and 8 post-intervention (Fig. 4 d). To investigate whether BH4 levels were increased under the reduction of nociceptive thresholds, the levels of the pterin were measured in the urine of the animals. Figure 4 e shows higher levels of BH4 in the urine of HFD-fed animals at 4 weeks [ t (5) = 3.73; P < 0.01], and 8 weeks [ t (4) = 3.32; P < 0.01] after the intervention. To verify whether the level of DNA methylation was responsible for the increased BH4 levels in the urine, the degree of methylation of the promoter of genes involved in BH4 biosynthesis was assessed. Figure 5 shows that the percentage of methylation of the promoters for Dhfr (Fig. 5 a), Spr (Fig. 5 b) and Ptps (Fig. 5 c) was not changed in mice receiving HFD. HFD-fed mice were also submitted to moderate-intensity physical exercise for 5 weeks, starting three weeks after initiating the dietary intervention. Supplementary Fig. 1a illustrates the results of the incremental test, represented by the percentage of success. Supplementary Fig. 1b shows increased lactate concentrations after the incremental test, demonstrating exhaustion. Figure 6 shows the effects of physical exercise on caloric intake, body weight and vWAT accumulation. As shown in Fig. 6 a caloric consumption was higher after 2 weeks of exercise, although no differences were observed in the percentage of weight gain (Fig. 6 b), and in the weight gain delta (Fig. 6 c). Physical exercise caused a decrease in vWAT weight [ t (11) = 2.56; P ≤ 0.05] (Fig. 6 d), and body fat [ t (11) = 2.64; P ≤ 0.05] (Fig. 6 e) after 6 weeks of the physical exercise intervention. Supplementary Figs. 3a and 3b are representative images from a mouse from each experimental group. Figure 7 shows the effects of physical exercise on GTT and nociceptive parameters after the interventions, diet plus exercise. Figure 7a shows that the GTT was significant decreased after 15 min of the glucose challenge in exercised mice submitted to HFD [ F (6,66) = 2.74; P ≤ 0.05]. Similarly, the area under the curve was also reduced [ t (11) = 2.41; P ≤ 0.05] ( Figure 7b ). However, fasting glucose ( Figure 7c ) was not different between groups at the end of the interventions. When the nociceptive thresholds were assessed, no differences were observed in ( Figure 7d ) chemical or thermal hyperalgesia ( Figure 7e ). However, mechanical hyperalgesia was reduced at week 6 ( Figure 7g ) in exercised mice submitted to HFD [ t (11) = 18.21; P < 0.01]. 4 Discussion Obesity is a chronic disease characterized by excessive accumulation of vWAT, in general associated to increased consumption of dietary fats and a lifestyle with more sedentary behaviour (WHO 2021 ). HFD-fed mice are commonly used to study potential treatments or the physiopathology of obesity because the induced metabolic alterations are similar to those seen in individuals affected by the disease (West and York 1998 ). Here, we showed that HFD promoted vWAT accumulation, increased vWAT to total body weight ratio, and increased body weight in adult male C57BL/J6 mice. The energy efficiency of the diets showed higher weight gain per calorie intake in HFD-fed mice. However, the weekly food consumption showed that, on average, 24 g of standard diet and 13 g of HFD were consumed, resulting in 50 kcal/g and 20 kcal/g respectively. That resulted in a lower caloric intake in HFD group. Obesity is evidenced by adiposity in visceral and subcutaneous WATs (West and York 1998 ). Considering that lipids are significant sources of energy and that adipocytes have great potential for hypertrophy and hyperplasia in response to fat ingestion, the saturated fat in HFD plays a fundamental role in the formation of large deposits of body fat (Townsend, Lorenzi, and Widmaier 2008). Our results are in agreement with other studies that fed mice with HFD and observed increased body weight gain, visceral fat accumulation and changes in the lipid profile (Atshaves et al. 2010 ). The adipose tissue is a complex, essential, and highly active metabolic and endocrine organ that constitutes a source of hormones, peptides, cytokines, and adipokines (Hotamisligil 2017 ). Adipocytes in WAT release MCP-1 (monocyte chemoattractant protein-1), which attracts monocytes and favors their differentiation into pro-inflammatory polarized M1 macrophages forming “crown-like structures” around the vWAT. This mechanism is known to provoke the dead of adipocytes and increased local levels of TNF-α, IL-1β, and IL-6 (Lumeng, Bodzin, and Saltiel 2007). Furthermore, adipocytes tend to rupture during obesity due to the limited capacity for expansion, leading to apoptosis, and consequently, to sustained inflammation. In this scenario, it is known that the pro-inflammatory IL-1β released by adipocytes can rapidly and directly activate nociceptors and induce hypersensitivity to pain (Binshtok et al. 2008 ). WAT is innervated by sensory neurons and presents bidirectional communication with the brain through afferent and efferent sensory fibers (Fishman and Dark 1987 ). Adipocyte size, lipid mobilization and paracrine secretion are controlled by WAT nerve endings (Bartness et al. 2014 ). Thus, sensory neurons that innervate the WAT are involved in the production of cytokines and the influx of immune cells, playing a central role in the low-grade inflammation observed in obesity (Bartness et al. 2014 ). The increase vWAT in mice fed with HFD that we observed plays an important role in the development of mechanical hypersensitivity. Mechanical hyperalgesia can be triggered by the process of hypertrophy and hyperplasia of adipocytes in an inflammatory state. IL-1β is also a stimulus that massively activates BH4 synthesis via the de novo pathway in immune cells, sensitizing nociceptive fibers and contributing to pain hypersensitivity (Cronin et al. 2018 ; Eichwald et al. 2023 ; Fujita et al. 2020 ; Latremoliere et al. 2015 ; Scheffer and Latini 2020; Werner et al. 1990 ). The relationship between BH4 and pain was first discovered, through the identification of an allele of the GCH1 gene haplotype that encodes for the GTP cyclohydrolase, the rate-limiting enzyme for BH4 biosynthesis, associated with reduced pain scores in multiple independent neuropathic pain cohorts (for review see Tegeder et al. 2006 ). Based in this human validation of the biological role of BH4 in neuropathic and inflammatory pain, we demonstrated that excessive BH4 levels were produced by neurons in active pain, and immune cells infiltrating damaged nerves and inflamed tissues (Cronin et al. 2018 ; Fujita et al. 2020 ; Latremoliere et al. 2015 ). Based on this information, two inhibitors for BH4 production were developed (SPRi3 and QM385) and showed that inflammation and pain hypersensitivity were reduced along with decreased BH4 levels in targeted tissues (Cronin et al. 2018 ; Fujita et al. 2020 ; Latremoliere et al. 2015 ). Furthermore, we discovered that the BH4-related metabolite, sepiapterin, accumulates in tissues and fluids exposed to the inhibitors (Fujita et al. 2020 ). Urinary sepiapterin was also validated as a sensitive, specific, and non-invasive biomarker in a cohort of healthy humans receiving sulfasalazine, a pharmacological treatment approved by the FDA for treating inflammatory bowel diseases. Indeed, sulfasalazine was recently described to be an inhibitor of BH4 synthesis (Chidley et al. 2011 ). These ground-breaking findings allowed us to hypothesize that excessive BH4 levels may play a significant role in pain development. The involvement of BH4 metabolism in animal models for inflammatory and neuropathic nociception showed a marked increase in BH4 synthesis in sensory neurons and nervous tissues inducing hyperalgesia (Latremoliere et al. 2015 ). The data presented here showed that increased WAT accumulation also favored the synthesis of BH4, and possibly, the development of the observed mechanical and chemical hyperalgesia. Increased transcription of GCH1 and higher levels of BH4 have previously been reported in leukocytes infiltrating injured sciatic nerves, reinforcing the contribution of the immune system in the induction and maintenance of pain induced by the pathological production of BH4 (Latremoliere et al. 2015 ). Furthermore, the present data are in agreement with we our previous report showing increased levels of plasma neopterin, the by-product of BH4 metabolism that normally increases proportionally to BH4, in individuals affected by type III obesity (Lenoir da Silva et al. 2017). BH4 is produced by macrophages that infiltrate tissues as a response to inflammation (Sakai, Kaufman, and Milstein 1993 ) and its overproduction correlates with hyperalgesia (Latremoliere et al. 2015 ). Thus, increased vWAT appears to be a favourable environment for the development of pain hypersensitivity. Indeed, the results demonstrated here showed concomitant increase in WAT, mechanical hyperalgesia, and BH4 levels. Furthermore, the urinary BH4 can be proposed as a potential easy-to-access, sensitive and reliable biomarker of pain development, and a promising target for the control of pain hypersensitivity in obesity. The use of pharmacological inhibitors aimed at reducing high levels of BH4 provide a potential therapeutic tool in the treatment of chronic pain. The literature has extensively demonstrated that the regular practice of moderate-intensity physical exercise is beneficial for health promotion, and for decreasing the risk of death from all causes (for a review see Bartness et al. 2014 ). The Physical Activity Guidelines for Americans points out that in individuals affected by chronic diseases, the practice of moderate-intensity physical exercise for 150 min a week, or the practice of high-intensity aerobic physical activity for 75 min a week, are beneficial in improving the health of chronic illnesses affected individuals. Also, muscle-strengthening activities involving all major muscle groups, if practiced at least twice a week, provide additional health benefits (U.S. Department of Health and Human Services 2018 ). The effect of this intervention is more pronounced in the conditions in which the physiopathology is associated with persistent activation of the immune system (Nieman and Wentz 2019). It is known that the regular practice of moderate-intensity physical exercise reduces systemic inflammation. Our group demonstrated that lipopolysaccharide-induced inflammation in mice provoked increased glycemia levels in the GTT and led to an increase in the concentrations of urinary neopterin (Scheffer and Latini 2020), a sensitive marker of immune system activation (Huber et al. 1984 ). We also showed that the GTT and neopterin levels were normalised by physical exercise (Scheffer et al. 2019 ). Additionally, we have also shown that neopterin is increased in the plasma of individuals affected by obesity type III (Lenoir da Silva et al. 2017), and insulin resistance-associated obesity, who also showed increased markers of inflammation (Remor et al. 2018 ). Additionally, we have also shown that exercise modulated the inflammatory response induced by lipopolysaccharide. This anti-inflammatory status was characterized by reduced levels of neopterin in the urine (Scheffer et al. 2019 ). Thus, regular practice of physical exercise positive modulates the anti-inflammatory response of the immune system normalizing the hypersensitivity scores seen, and possibly, the pathological production of BH4, in mice with increased vWAT. 5 Conclusions This study showed that increased vWAT elicited the overproduction and secretion of BH4 with consequent increased scores of chemical and mechanical hyperalgesias. Thus, urinary BH4 can be proposed as a biomarker of pain in obesity that is easy-to-access and no invasive. Finally, physical exercise can prevent the development of metabolic diseases and chronic pain, possibly by reducing WAT accumulation, BH4 overproduction, and therefore the associated inflammatory state. 6 Limitations of the study Using male mouse restricted our results since males and females often exhibit differences in metabolic composition due to the influence of sex hormones and various genetic and physiological factors. Male mice typically have a higher lean body mass, which can affect their metabolic rate, leading to variations in energy expenditure, nutrient utilization, and fat distribution. Declarations 7 Funding This research was supported by the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil; CNPq Universal #422488/2021-6). A.L. is a CNPq fellow (CNPq PQ #312854/2019-6). 8 Acknowledgments We thank the LAMEB/CCB-UFSC technicians for their assistance, and Ted Griswold for the English editing. 9 Conflicts of Interest The authors declare no conflicts of interest. Author Contribution AL conceived the study with inputs from JMG and TE. AL and TE wrote the manuscript. TE, LB, AFS, DS, ACSP and JMG performed and designed the experimental strategy. AL and TE performed the statistical analysis and prepared all the figures. VM, ACS, CSS, RFS, MFR commented and edited the final version of the manuscript. References Aguiar, A. S., E. L. G. Moreira, A. A. Hoeller, P. A. 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Woolf, Clifford J., and Qiufu Ma. 2007. “Nociceptors—Noxious Stimulus Detectors.” Neuron 55(3):353–64. doi: 10.1016/j.neuron.2007.07.016. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx 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-4458806","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309676672,"identity":"ae588955-e0dd-44b5-90b8-6359078c80df","order_by":0,"name":"Tuany Eichwald","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Tuany","middleName":"","lastName":"Eichwald","suffix":""},{"id":309676673,"identity":"6722b1db-96c6-4b40-8247-5aee0af47939","order_by":1,"name":"Leonardo Barros","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Leonardo","middleName":"","lastName":"Barros","suffix":""},{"id":309676674,"identity":"68356499-f371-4552-80c0-96530fc0986d","order_by":2,"name":"Alexandre Francisco Solano","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Alexandre","middleName":"Francisco","lastName":"Solano","suffix":""},{"id":309676675,"identity":"554f1b85-8122-435b-913f-dadf8be8d6cb","order_by":3,"name":"Débora da Luz Scheffer","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Débora","middleName":"da Luz","lastName":"Scheffer","suffix":""},{"id":309676676,"identity":"9c359f14-fbff-4509-836c-c1c7dfea7e7b","order_by":4,"name":"Vivian Menegassi","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Vivian","middleName":"","lastName":"Menegassi","suffix":""},{"id":309676677,"identity":"16092176-e3ce-43d4-b3c1-61824ced9882","order_by":5,"name":"Ananda Christina Staats Pires","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Ananda","middleName":"Christina Staats","lastName":"Pires","suffix":""},{"id":309676678,"identity":"9a33abad-4e14-4c48-af07-94deba5dfacc","order_by":6,"name":"Camila Sartor Spivakoski","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Camila","middleName":"Sartor","lastName":"Spivakoski","suffix":""},{"id":309676679,"identity":"98cd5b06-d992-4fba-b8ba-9125f1ad7931","order_by":7,"name":"Rodrigo A. 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Controls received a standard rodent diet for the same period (20 kcal % protein, 70 kcal % carbohydrates and 10 kcal % lipids). (\u003cstrong\u003ea\u003c/strong\u003e) Daily calorie intake. (\u003cstrong\u003eb\u003c/strong\u003e) Delta of weight gain between final weight and initial weight. (\u003cstrong\u003ec\u003c/strong\u003e) Energy efficiency calculated by dividing the energy content (in kcal) by weight of foods (in g) consumed. (\u003cstrong\u003ed\u003c/strong\u003e) Accumulated visceral WAT after 8 weeks of intervention. (\u003cstrong\u003ee\u003c/strong\u003e) Relationship between visceral WAT and body weight. ANOVA for repeated measures followed by Šídák \u003cem\u003epost hoc\u003c/em\u003e test for multiple comparisons for \u003cstrong\u003ea\u003c/strong\u003e. One tail Student’s \u003cem\u003et\u003c/em\u003e-test for B, C, D, and E. * \u003cem\u003eP\u003c/em\u003e ≤ 0.05; ** \u003cem\u003eP\u003c/em\u003e ≤ 0.01; *** \u003cem\u003eP\u003c/em\u003e ≤ 0.001 (n= 6-7 animals per group).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4458806/v1/149a1f3bf8765b601ad95b70.jpg"},{"id":57699265,"identity":"f8e92ec8-f65e-4e43-8b2b-46c560a6f38a","added_by":"auto","created_at":"2024-06-04 13:30:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":134272,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlucose tolerance test (GTT) impairment in C57BL/J6 mice submitted to high fat diet (HFD).\u003c/strong\u003e Adult male C57BL/J6 mice received HFD \u003cem\u003ead libitum\u003c/em\u003efor 8 consecutive weeks (20 kcal % protein, 35 kcal % carbohydrates and 45 kcal % lipids). Controls received a standard diet for the same period (20 kcal % protein, 70 kcal % carbohydrates and 10 kcal % lipids). (\u003cstrong\u003ea,d,g\u003c/strong\u003e) Glucose tolerance test (GTT), (\u003cstrong\u003eb,e,h\u003c/strong\u003e) area under the curve of the GTT, and (\u003cstrong\u003ec,f,i\u003c/strong\u003e) fasting glucose were measured before the intervention, and at 4 and 8 weeks afterwards, respectively. ANOVA for repeated measures followed by Šídák \u003cem\u003epost hoc\u003c/em\u003e test for multiple comparisons for \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003eg\u003c/strong\u003e. One tail Student’s \u003cem\u003et\u003c/em\u003e-test for \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, and \u003cstrong\u003ei\u003c/strong\u003e. * \u003cem\u003eP\u003c/em\u003e ≤ 0.05; ** \u003cem\u003eP\u003c/em\u003e ≤ 0.01; *** \u003cem\u003eP\u003c/em\u003e ≤ 0.001 (n= 6-7 animals per group).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4458806/v1/acc691754f8b7655bd3ed2f8.jpg"},{"id":57700020,"identity":"1a662158-5997-4fb3-91c6-acaa65505107","added_by":"auto","created_at":"2024-06-04 13:38:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":134121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMotivational behavior and spontaneous locomotor activity were not altered in C57BL/J6 mice submitted to high fat diet (HFD).\u003c/strong\u003e Adult male C57BL/J6 mice received HFD \u003cem\u003ead libitum\u003c/em\u003e for 8 consecutive weeks (20 kcal % protein, 35 kcal % carbohydrates and 45 kcal % lipids). Controls received a standard rodent diet for the same period (20 kcal % protein, 70 kcal % carbohydrates and 10 kcal % lipids). (\u003cstrong\u003ea\u003c/strong\u003e) Latency, (\u003cstrong\u003eb\u003c/strong\u003e) total time and (\u003cstrong\u003ec\u003c/strong\u003e) number of grooming evoked by spraying sucrose on the back. (\u003cstrong\u003ed\u003c/strong\u003e) Total distance traveled, (\u003cstrong\u003ee\u003c/strong\u003e) number of intersections, (\u003cstrong\u003ef\u003c/strong\u003e) average speed, (\u003cstrong\u003eg\u003c/strong\u003e) maximum speed, (\u003cstrong\u003eh\u003c/strong\u003e) time spent in the center of the apparatus, and (\u003cstrong\u003ei\u003c/strong\u003e) time spent on the periphery of the apparatus. One-way ANOVA followed by Tukey \u003cem\u003epost hoc\u003c/em\u003e test. * \u003cem\u003eP\u003c/em\u003e ≤ 0.05 (n= 5-11 animals per group).\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4458806/v1/6873aadad0f224ea3751cee2.jpg"},{"id":57699258,"identity":"10648bdc-f75e-4ace-afd1-d4da07be34bb","added_by":"auto","created_at":"2024-06-04 13:30:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":95477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInduction of hyperalgesia and increased urinary tetrahydrobiopterin (BH4) levels in C57BL/J6 mice submitted to high fat diet (HFD).\u003c/strong\u003e Adult male C57BL/J6 mice received HFD \u003cem\u003ead libitum\u003c/em\u003e for 8 consecutive weeks (20 kcal % protein, 35 kcal % carbohydrates and 45 kcal % lipids). Controls received a standard diet for the same period (20 kcal % protein, 70 kcal % carbohydrates and 10 kcal % lipids). (\u003cstrong\u003ea\u003c/strong\u003e) First response latency, and (\u003cstrong\u003eb\u003c/strong\u003e) total response time evoked by hind paw subcutaneous injection of capsaicin. (\u003cstrong\u003ec\u003c/strong\u003e) Latency for the first response evoked by the heat of the hot plate at 50 ºC. (\u003cstrong\u003ed\u003c/strong\u003e) 50 % of the von Frey threshold used to determine mechanical hyperalgesia evaluated before, and at weeks 2, 4, 6 and 8 after the intervention. (\u003cstrong\u003ee\u003c/strong\u003e) BH4 concentrations in the urine before, and at 4 and 8 weeks after the intervention. ANOVA for repeated measures followed by Šídák \u003cem\u003epost hoc\u003c/em\u003etest for multiple comparisons for \u003cstrong\u003ed\u003c/strong\u003e. One tail Student’s \u003cem\u003et\u003c/em\u003e-test for \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, and \u003cstrong\u003ee\u003c/strong\u003e. * \u003cem\u003eP\u003c/em\u003e ≤ 0.05; ** \u003cem\u003eP\u003c/em\u003e ≤ 0.01 (n= 5-7 animals per group).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4458806/v1/f57ee338b3687158c8d6ec5b.jpg"},{"id":57700021,"identity":"97e55cd0-a635-4131-80d3-78cda8d5b28d","added_by":"auto","created_at":"2024-06-04 13:38:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNA methylation of the promoters for genes involved in tetrahydrobiopterin (BH4) biosynthesis was not altered in C57BL/J6 mice submitted to high fat diet (HFD).\u003c/strong\u003e Adult male C57BL/J6 mice received HFD \u003cem\u003ead libitum\u003c/em\u003e for 8 consecutive weeks (20 kcal % protein, 35 kcal % carbohydrates and 45 kcal % lipids). Controls a standard rodent diet for the same period (20 kcal % protein, 70 kcal % carbohydrates and 10 kcal % lipids).\u003cstrong\u003e \u003c/strong\u003eThe percentage of promoter methylation for (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eDhfr\u003c/em\u003e, (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eSpr\u003c/em\u003eand (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003ePtps\u003c/em\u003e were measured after 8 weeks HFD. One tail Student’s \u003cem\u003et\u003c/em\u003e-test (n=4 animals per group).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4458806/v1/f7539086acb86d460a96b77f.jpg"},{"id":57699260,"identity":"21104218-2901-45f5-86b9-2bbface3f1f3","added_by":"auto","created_at":"2024-06-04 13:30:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":108235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysical exercise reduced visceral white adipose tissue (WAT) and body fat in C57BL/J6 mice submitted to high fat diet (HFD).\u003c/strong\u003e Adult male C57BL/J6 mice received HFD \u003cem\u003ead libitum\u003c/em\u003e for 8 consecutive weeks (20 kcal % protein, 35 kcal % carbohydrates and 45 kcal % lipids). After 2 weeks of the dietary intervention, a group of mice were also submitted to physical exercise 5 times per week for 6 weeks (for details see M\u0026amp;M). (\u003cstrong\u003ea\u003c/strong\u003e) Calorie intake, and (\u003cstrong\u003eb\u003c/strong\u003e) body weight gain were assessed daily for the first week of the treatment, and weekly for the next 7 weeks. (\u003cstrong\u003ec\u003c/strong\u003e) Delta of weight gain between final weight and initial weight. (\u003cstrong\u003ed\u003c/strong\u003e) Visceral WAT in grams after 8 weeks of interventions (8 weeks of dietary intervention plus 6 weeks of concomitant physical exercise). (\u003cstrong\u003ee\u003c/strong\u003e) Relationship between visceral WAT and body weight. ANOVA for repeated measures followed by Šídák \u003cem\u003epost hoc\u003c/em\u003e test for multiple comparisons for \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eb\u003c/strong\u003e. One tail Student’s \u003cem\u003et\u003c/em\u003e-test for \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, and \u003cstrong\u003ee\u003c/strong\u003e. * \u003cem\u003eP\u003c/em\u003e ≤ 0.05; ** \u003cem\u003eP\u003c/em\u003e ≤ 0.01; *** \u003cem\u003eP\u003c/em\u003e ≤ 0.001 (n= 6-7 animals per group).\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4458806/v1/499dd1fbd587d8f300db4453.jpg"},{"id":57699262,"identity":"f1706bb6-15b7-467c-9b40-d0116dde7173","added_by":"auto","created_at":"2024-06-04 13:30:32","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":120237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysical exercise normalized the glucose tolerance test and hyperalgesia evoked by mechanical stimuli in C57BL/J6 mice submitted to high fat diet (HFD).\u003c/strong\u003e Adult male C57BL/J6 mice received HFD \u003cem\u003ead libitum\u003c/em\u003e for 8 consecutive weeks (20 kcal % protein, 35 kcal % carbohydrates and 45 kcal % lipids). After 2 weeks of the dietary intervention, mice were also submitted to physical exercise 5 times per week for 6 weeks (for details see M\u0026amp;M). (\u003cstrong\u003ea\u003c/strong\u003e) Glucose tolerance test (GTT), (\u003cstrong\u003eb\u003c/strong\u003e) area under the curve of the GTT, and (\u003cstrong\u003ec\u003c/strong\u003e) fasting glucose were measured after the interventions (week 8). (\u003cstrong\u003ed\u003c/strong\u003e) First response latency, and (\u003cstrong\u003ee\u003c/strong\u003e) total response time evoked by an injection of capsaicin in the right paw (for details see M\u0026amp;M). (\u003cstrong\u003ef\u003c/strong\u003e) Latency for the first response evoked by the heat of the hot plate at 50 ºC. (\u003cstrong\u003eg\u003c/strong\u003e) 50 % of the von Frey threshold used to determine mechanical hyperalgesia evaluated before, and at weeks 2, 4, 6 and 8 after the interventions. ANOVA for repeated measures followed by Šídák \u003cem\u003epost hoc\u003c/em\u003e test for multiple comparisons for \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eg\u003c/strong\u003e. One tail Student’s \u003cem\u003et\u003c/em\u003e-test for \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, and \u003cstrong\u003ef\u003c/strong\u003e. * \u003cem\u003eP\u003c/em\u003e ≤ 0.05; ** \u003cem\u003eP\u003c/em\u003e ≤ 0.01 (n= 6-7 animals per group).\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4458806/v1/aa207d9c9f4a59025d832f7e.jpg"},{"id":59750195,"identity":"1f5d68b5-7388-4df6-b333-38643b77c98c","added_by":"auto","created_at":"2024-07-05 20:03:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1757293,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4458806/v1/b085a252-7747-49f4-bff3-94323f153f1d.pdf"},{"id":57699263,"identity":"aedacb7a-dbd2-4f51-aaa9-92ab22a4adfb","added_by":"auto","created_at":"2024-06-04 13:30:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":238406,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4458806/v1/89e991302647d27e67b59835.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pain hypersensitivity and increased urinary tetrahydrobiopterin levels in mice submitted to high fat diet","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eObesity is defined by the World Health Organization (WHO) as abnormal and excessive accumulation of fat (WHO \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The increase in white adipose tissue (WAT) from a daily positive energy balance is associated with low-grade chronic systemic inflammation, with immune infiltration in the WAT. Obesity is a multifactorial etiology disease, and the clinical manifestations involve chronic pain and joint dysfunction (Thomazeau et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Obesity predisposes the development of a large number of chronic diseases, including inflammatory and degenerative conditions of the musculoskeletal system and the peripheral and central nervous systems (Cope et al. 2018; Smith et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Indeed, the inflammatory pathways are persistently activated in several brain regions controlling peripheral regulation of energy, glucose, and lipid metabolism (Gaspar et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Hotamisligil \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTetrahydrobiopterin (BH4) is a pterin that acts biologically as an mandatory cofactor for the metabolism of phenylalanine and certain lipids, and for the biosynthesis of the neurotransmitters dopamine, serotonin and nitric oxide (NO) (Werner et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Werner, Blau, and Th\u0026ouml;ny \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). BH4 intracellular concentrations are finely tuned by three metabolic pathways, assuring continue basal levels of the molecule to supports the systems where it is involved (see Eichwald et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e for a review). However, excessively increased BH4 levels have been associated with numerous pathological conditions, including cardiovascular disease (Bendall et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), cancer (Cronin et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and chronic pain (Latremoliere et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tegeder et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Furthermore, inflammatory conditions positively modulate the synthesis of BH4, in immune, nerve cells, and others (Ghisoni et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; de Paula Martins et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePain is a conscious experience that demands cortical participation and aversive information from nociception, processed by the peripheral nervous system, and unconsciously modulated by the central nervous system (Woolf and Ma 2007). Normally, the adaptive response against the stimuli that activate both systems overcome the threat and reach the resolution. However, maladaptive inflammatory reactions, in which pro-inflammatory mediators persistently activate and sensitize neurons at different levels of the nociceptive pathway, are believed to induce chronic pain (Costigan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Eichwald and Talbot 2020). Thus, this work aimed to identify whether obesity, a low-degree chronic inflammatory disease, may negatively modulate nociceptive thresholds and positively increase BH4 levels that can be monitored in biological fluids as a biomarker for pain.\u003c/p\u003e"},{"header":"2 Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Animals\u003c/h2\u003e\n \u003cp\u003eAdult male C57BL/J6 mice (3\u0026ndash;5 months of age; 45\u0026ndash;50 g) from the Centre for Biological Sciences, \u003cem\u003eUniversidade Federal de Santa Catarina\u003c/em\u003e (UFSC) (Brazil) were acclimated for ten days in a controlled environment (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 12 h light/dark cycle) with free access to water and food. All experimental protocols, approved by UFSC\u0026rsquo;s Ethics Committee for Animal Research (CEUA, 4401201118), complied with current guidelines for laboratory animals and ethical care and ethical standards for experimental pain research in conscious animals (Percie du Sert et al. 2020).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Experimental strategy\u003c/h2\u003e\n \u003cp\u003eMice were randomly divided into two groups. One group received a high-fat diet (HFD), while the other group received a standard diet. Both groups were maintained on their respective diets for 8 weeks. Mice were fed with HFD (n\u0026thinsp;=\u0026thinsp;26) to induce increased adiposity, and to be used as a proxy of human obesity. The diet\u0026rsquo;s macronutrient composition was proteins: 20 kcal %; carbohydrates: 35 kcal %; lipids: 45 kcal % (Pragsolu\u0026ccedil;\u0026otilde;es Bioci\u0026ecirc;ncias, Ja\u0026uacute;, S\u0026atilde;o Paulo, Brazil). Animals fed with standard diet were used as controls (n\u0026thinsp;=\u0026thinsp;12). The standard diet composition was protein: 20 kcal %; carbohydrates: 70 kcal %; lipids: 10 kcal %. The animals were housed in cages containing 4\u0026ndash;5 individuals from the same experimental group. Cages were labeled with the type of treatment (diets), and the corresponding food was provided. During the whole experimental strategy, mice were closely monitored for humane endpoints.\u003c/p\u003e\n \u003cp\u003eExperimental groups consisted of \u003cstrong\u003en\u0026thinsp;=\u0026thinsp;6\u0026ndash;7\u003c/strong\u003e mice. This number allowed to perform the behavioral tests with sensitivity and reproducibility and to collect tissues generating an appropriate unit of mass to perform the biochemical measurements. This number of sample size was calculated by applying \u0026ldquo;power statistics\u0026rdquo;, as shown below.\u003c/p\u003e\n \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSample size calculation\u003c/span\u003e: We used the following formula for sample size calculation for the comparison between two groups with quantitative data endpoints: Sample size\u0026thinsp;=\u0026thinsp;2 SD\u003csup\u003e2\u003c/sup\u003e (Z\u003csup\u003ea/2\u003c/sup\u003e + Z\u003csup\u003e\u0026beta;\u003c/sup\u003e)\u003csup\u003e2\u003c/sup\u003e/d\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eWhere: Standard deviation (SD)\u0026thinsp;=\u0026thinsp;estimated from previous studies\u003c/p\u003e\n \u003cp\u003eZ\u003csup\u003ea/2\u003c/sup\u003e = Z \u003csub\u003e0.05/2\u003c/sub\u003e = Z \u003csub\u003e0.025\u003c/sub\u003e = 1.96 (From Z table) at type 1 error of 5%\u003c/p\u003e\n \u003cp\u003eZ\u003csup\u003e\u0026beta;\u003c/sup\u003e = Z 0.20\u0026thinsp;=\u0026thinsp;0.842 (From Z table) at 80% power\u003c/p\u003e\n \u003cp\u003ed\u0026thinsp;=\u0026thinsp;effect size\u0026thinsp;=\u0026thinsp;difference between mean values\u003c/p\u003e\n \u003cp\u003eHence now formula will be:\u003c/p\u003e\n \u003cp\u003eSample size\u0026thinsp;=\u0026thinsp;2 SD\u003csup\u003e2\u003c/sup\u003e (1.96\u0026thinsp;+\u0026thinsp;0.842)\u003csup\u003e2\u003c/sup\u003e/d\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003eBased on previous studies from our group we have the following assumptions:\u003c/p\u003e\n \u003cp\u003e- The minimum difference between groups\u0026rsquo; mean will be set at least at 54.4%;\u003c/p\u003e\n \u003cp\u003e- Biological experiments inherently have 10\u0026ndash;15% error margin;\u003c/p\u003e\n \u003cp\u003e- Differences less than 20% of each group\u0026rsquo;s mean can increase the probability of type I or type II errors;\u003c/p\u003e\n \u003cp\u003e- The standard deviation is typically 35% of the mean value.\u003c/p\u003e\n \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSample size\u003c/span\u003e\u0026thinsp;=\u0026thinsp;2 35\u003csup\u003e2\u003c/sup\u003e (1.96\u0026thinsp;+\u0026thinsp;0.842)\u003csup\u003e2\u003c/sup\u003e/54.4\u003csup\u003e2\u003c/sup\u003e= \u003cstrong\u003e6.5.\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Intraperitoneal glucose tolerance test\u003c/h2\u003e\n \u003cp\u003eThe glucose tolerance test (GTT) was performed after 6 h of fasting by injecting mice with 2 g/kg glucose intraperitoneally (i.p.). Blood glucose levels were measured at 0, 5, 10, 15, 30, 60 and 120 min from tail blood samples (adapted from Rafacho et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e). The test was performed before the dietary intervention, at 4 and 8 weeks.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Physical exercise\u003c/h2\u003e\n \u003cp\u003eTo prevent HFD-induced WAT accumulation, a group of animals was also submitted to physical exercise as described below.\u003c/p\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.1 Incremental test to determine the maximal capacity for exercise\u003c/h2\u003e\n \u003cp\u003eThe incremental test was performed to identify 60% of the maximal capacity of the animals (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;1a\u003c/strong\u003e) (Aguiar et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). When exhaustion was reached, caudal blood was collected to measure lactate levels.\u003c/p\u003e\n \u003cp\u003eDuring the first week of training, performance was scored daily: 1 for animals that refused to run; 2 for animals that ran at variable speeds, run, and stop; 3 for animals that ran regularly; 4 for animals that are runners; 5 for animals that are good runners. Only animals that scored 3 or more continued the training. Two animals scored less than 3 and were discontinued from the physical exercise protocol.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.2 Physical exercise protocol\u003c/h2\u003e\n \u003cp\u003eThe physical exercise protocol consisted of five training sessions/week for six weeks on a treadmill with an interval of 48 h each week (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) (Aguiar et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Animals began with a 5 min warm-up at 40% intensity before each session. Training speed was set at 60% of the final speed from the incremental test, indicating moderate intensity. Sessions lasted 35, 40, and 45 min/day for the first 3 weeks, with a subsequent incremental test. The last 2 weeks sessions lasted 35 and 40 min/day.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhysical exercise protocol\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTraining weeks\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSession duration (min)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreadmill inclination (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eActivity intensity (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eEffort test to equalize intensities\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv id=\"Sec9\" class=\"Section4\"\u003e\n \u003ch2\u003e2.4.2.1 Caudal blood collection\u003c/h2\u003e\n \u003cp\u003eImmediately at the end of the incremental test, caudal blood was collected in tubes containing sodium fluoride to inhibit the glycolysis (Rhoden and Rhoden 2006). Blood was used to measure lactate concentrations.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e\n \u003ch2\u003e2.4.2.2 Lactate measurement\u003c/h2\u003e\n \u003cp\u003eThe specific analyzer YSL 2700 (YSL 2700, Yellow Springs, CA, USA) was used to quantify blood lactate levels. High blood lactate indicated exercise protocol\u0026rsquo;s intensity.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Behavioral tests\u003c/h2\u003e\n \u003cp\u003eAnimals were acclimated for 1 h before behavioral tasks in the experimental room. Assessments occurred during the rodent\u0026apos;s light phase and were conducted by a researcher blinded to the experimental groups.\u003c/p\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.1 Locomotor activity\u003c/h2\u003e\n \u003cp\u003eLocomotor activity was evaluated in a 100 cm\u0026times;100 cm\u0026times;50 cm open field arena in a sound-attenuated room under low-intensity light. Each animal\u0026rsquo;s exploratory activity was video recorded for 5 min, and analyzed using the ANY-mazy Platform\u0026trade; (Aguiar et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). Animals were evaluated before and after the dietary intervention.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.2 Motivational behavior\u003c/h2\u003e\n \u003cp\u003eMotivational behavior was assessed by measuring for 5 min the grooming behavior after mice being squirted with 1 mL of a 10% sucrose solution on their dorsal coat (Scheffer et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Animals were assessed before and after the dietary intervention.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.3 Mechanical hypersensitivity\u003c/h2\u003e\n \u003cp\u003eTo assess mechanical sensitivity, the withdrawal threshold was measured using a series of von Frey filaments (0.20, 0.40, 0.70, 1.6, 3.9, 5.9, 9.8 and 13.7 mN, Stoelting, Wood Dale, IL, USA; equivalent in grams to 0.02, 0.04, 0.07, 0.16, 0.40, 0.60, 1.0 and 1.4). The 50% withdrawal threshold was determined using the \u0026lsquo;up-down\u0026rsquo; method and calculated using Up-Down Reader software (Gonzalez-Cano et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Animals were evaluated before and 2, 4, 6, and 8 weeks after the dietary intervention.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.4 Thermal hypersensitivity\u003c/h2\u003e\n \u003cp\u003eMice were placed in an acrylic cylinder on the surface of a previously heated metal plate (50\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) (INSIGHT\u0026reg;). The latency that the animal took to stand up, shake and/or lick one of the hind legs was considered as an indication of thermal hypersensitivity. Animals were evaluated after 8 weeks of dietary intervention.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.5 Chemical hypersensitivity\u003c/h2\u003e\n \u003cp\u003eAnimals received 20 uL injection containing 1.6 \u0026micro;g capsaicin under the skin of the dorsal right hind paw. The animal was then placed in a transparent glass chamber and observed for 5 min. The latency and the number of times of licking and/or shaking the paw, fingers, or leg where the capsaicin was injected was timed (Sakurada et al. \u003cspan class=\"CitationRef\"\u003e1992\u003c/span\u003e). Animals were assessed after the dietary intervention.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Adipose tissue dissection\u003c/h2\u003e\n \u003cp\u003eAnimals were euthanized at the end of 8 weeks of dietary intervention, following the ARRIVE guidelines, to collect blood and visceral WAT (vWAT).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 BH4 quantification\u003c/h2\u003e\n \u003cp\u003eUrinary BH4 levels were determined by high-performance liquid chromatography (HPLC) (Alliance e2695, Waters, MA, USA) coupled with electrochemical detection as previously described with some modifications (Latremoliere et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). The results were expressed as \u0026micro;mol/mmol of creatinine.\u003c/p\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e2.7.1 Quantification of creatinine\u003c/h2\u003e\n \u003cp\u003eUrinary creatinine concentrations were determined using a commercial kit (Pointe Scientific Inc., Canton, Michigan, USA). Creatinine levels were indicated as mmol/L.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean. Data were analyzed by one-way or two-way ANOVA followed by the \u003cem\u003epost hoc\u003c/em\u003e test of \u0026Scaron;\u0026iacute;d\u0026aacute;k when \u003cem\u003eF\u003c/em\u003e was significant. When comparing two independent groups, one tail Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test was used. The accepted level of significance for the tests was \u003cem\u003eP\u003c/em\u003e \u0026le; 0.05. Statistics and all graphs were performed by using GraphPad Prism 9\u0026reg;.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003eFigure 1 shows the effect of HFD on caloric consumption, body weight and fat accumulation. \u003cstrong\u003eFigure 1a\u0026nbsp;\u003c/strong\u003eshows that caloric consumption was significantly lower in animals submitted to HFD. However, \u003cstrong\u003eFigure 1b\u003c/strong\u003e shows that the weight gain delta, calculated as the final body weight minus the initial body weight, was increased in HFD fed mice [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e= 1.77; \u003cem\u003eP\u003c/em\u003e \u0026le; 0.05]. In agreement, the energy efficiency of the diets (\u003cstrong\u003eFigure 1c\u003c/strong\u003e) was shown to be higher in HFD-fed mice [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e= 2.28; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05]. \u003cstrong\u003eFigure 1d\u003c/strong\u003e shows that the weight of the vWAT was significantly higher in HFD-fed animals [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e= 3.38; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01]. Similarly, the relationship between vWAT and total body weight was increased in the group of animals fed with HFD (\u003cstrong\u003eFigure 1e\u003c/strong\u003e) [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e= 4.42; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001]. \u003cstrong\u003eSupplementary Figures 2a\u0026nbsp;\u003c/strong\u003eand\u0026nbsp;\u003cstrong\u003e2b\u003c/strong\u003e are representative images from a mouse from each experimental group.\u003c/p\u003e\u003cp\u003eGTT and the fasting glucose test were performed at three different times, baseline (pre-intervention), 4 and 8 weeks after intervention to identify insulin resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea shows that GTT was identical in both experimental groups before the dietary intervention. The area under the curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and fasting glucose levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) were also not different. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed shows that GTT was not altered after 4 weeks of intervention; however, the area under the curve shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee was significantly bigger in the HFD group [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.90; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001], as well as fasting blood glucose levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.903; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05]. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg shows significant increase in glycemia at 15 min [F\u003csub\u003e(6,66)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.68; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001], and the values under the curve [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.774; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh) after the glucose injection in HFD-fed mice. However, fasting glycemia at week 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei) was not different between groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the influence of HFD on motivational behavior and spontaneous locomotor activity. Figures\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c show that the performance in the sucrose test, used to assess motivation, depression, and anhedonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), and total time spent in grooming (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) were not altered in HFD-fed mice. However, grooming total time spent was longer in the group that received the standard diet after 8 weeks of intervention [\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,20)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.80; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). To identify whether the diet would cause changes in locomotor activity, the open field test was performed before the intervention and at the end of 8 weeks of experimentation. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh shows that animals fed with HFD for 8 weeks spent less time in the center of the apparatus [\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(2,23)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.11; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05]. No significant differences were observed between groups in total distance traveled (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), crossing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), average speed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), maximal speed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg), and time in the periphery (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether the HFD would induce nociceptive changes under different noxious stimuli, mice were evaluated for chemical, thermal and mechanical hyperalgesia. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea shows that there was no difference between groups in the latency for the animals' first response evoked by capsaicin. However, total response time evoked by capsaicin was higher in animals fed with HFD [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(9)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.11; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec shows that there was no significant difference in the reflexes evoked by the thermal stimulus. HFD-fed mice showed lower thresholds for mechanical hyperalgesia at weeks 2, 6 and 8 post-intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). To investigate whether BH4 levels were increased under the reduction of nociceptive thresholds, the levels of the pterin were measured in the urine of the animals. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee shows higher levels of BH4 in the urine of HFD-fed animals at 4 weeks [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(5)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.73; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01], and 8 weeks [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(4)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.32; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01] after the intervention.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify whether the level of DNA methylation was responsible for the increased BH4 levels in the urine, the degree of methylation of the promoter of genes involved in BH4 biosynthesis was assessed. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows that the percentage of methylation of the promoters for \u003cem\u003eDhfr\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), \u003cem\u003eSpr\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) and \u003cem\u003ePtps\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) was not changed in mice receiving HFD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHFD-fed mice were also submitted to moderate-intensity physical exercise for 5 weeks, starting three weeks after initiating the dietary intervention. \u003cb\u003eSupplementary Fig.\u0026nbsp;1a\u003c/b\u003e illustrates the results of the incremental test, represented by the percentage of success. \u003cb\u003eSupplementary Fig.\u0026nbsp;1b\u003c/b\u003e shows increased lactate concentrations after the incremental test, demonstrating exhaustion. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the effects of physical exercise on caloric intake, body weight and vWAT accumulation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea caloric consumption was higher after 2 weeks of exercise, although no differences were observed in the percentage of weight gain (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), and in the weight gain delta (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Physical exercise caused a decrease in vWAT weight [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.56; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), and body fat [\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.64; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee) after 6 weeks of the physical exercise intervention. \u003cb\u003eSupplementary Figs.\u0026nbsp;3a and 3b\u003c/b\u003e are representative images from a mouse from each experimental group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 7\u0026nbsp;\u003c/strong\u003eshows the effects of physical exercise on GTT and nociceptive parameters after the interventions, diet plus exercise.\u0026nbsp;\u003cstrong\u003eFigure 7a\u003c/strong\u003e shows that the GTT was significant decreased after 15 min of the glucose challenge in exercised mice submitted to HFD\u0026nbsp;[\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(6,66)\u003c/sub\u003e= 2.74; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026le; 0.05]. Similarly, the area under the curve was also reduced\u0026nbsp;[\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e= 2.41; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026le; 0.05]\u0026nbsp;(\u003cstrong\u003eFigure 7b\u003c/strong\u003e). However, fasting glucose (\u003cstrong\u003eFigure 7c\u003c/strong\u003e) was not different between groups at the end of the interventions. When the nociceptive thresholds were assessed, no differences were observed in (\u003cstrong\u003eFigure 7d\u003c/strong\u003e) chemical or thermal hyperalgesia (\u003cstrong\u003eFigure 7e\u003c/strong\u003e). However, mechanical hyperalgesia was reduced at week 6 (\u003cstrong\u003eFigure 7g\u003c/strong\u003e) in exercised mice submitted to HFD\u0026nbsp;[\u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e= 18.21; \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01].\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eObesity is a chronic disease characterized by excessive accumulation of vWAT, in general associated to increased consumption of dietary fats and a lifestyle with more sedentary behaviour (WHO \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). HFD-fed mice are commonly used to study potential treatments or the physiopathology of obesity because the induced metabolic alterations are similar to those seen in individuals affected by the disease (West and York \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Here, we showed that HFD promoted vWAT accumulation, increased vWAT to total body weight ratio, and increased body weight in adult male C57BL/J6 mice. The energy efficiency of the diets showed higher weight gain per calorie intake in HFD-fed mice. However, the weekly food consumption showed that, on average, 24 g of standard diet and 13 g of HFD were consumed, resulting in 50 kcal/g and 20 kcal/g respectively. That resulted in a lower caloric intake in HFD group.\u003c/p\u003e \u003cp\u003eObesity is evidenced by adiposity in visceral and subcutaneous WATs (West and York \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Considering that lipids are significant sources of energy and that adipocytes have great potential for hypertrophy and hyperplasia in response to fat ingestion, the saturated fat in HFD plays a fundamental role in the formation of large deposits of body fat (Townsend, Lorenzi, and Widmaier 2008). Our results are in agreement with other studies that fed mice with HFD and observed increased body weight gain, visceral fat accumulation and changes in the lipid profile (Atshaves et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe adipose tissue is a complex, essential, and highly active metabolic and endocrine organ that constitutes a source of hormones, peptides, cytokines, and adipokines (Hotamisligil \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Adipocytes in WAT release MCP-1 (monocyte chemoattractant protein-1), which attracts monocytes and favors their differentiation into pro-inflammatory polarized M1 macrophages forming \u0026ldquo;crown-like structures\u0026rdquo; around the vWAT. This mechanism is known to provoke the dead of adipocytes and increased local levels of TNF-α, IL-1β, and IL-6 (Lumeng, Bodzin, and Saltiel 2007). Furthermore, adipocytes tend to rupture during obesity due to the limited capacity for expansion, leading to apoptosis, and consequently, to sustained inflammation. In this scenario, it is known that the pro-inflammatory IL-1β released by adipocytes can rapidly and directly activate nociceptors and induce hypersensitivity to pain (Binshtok et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). WAT is innervated by sensory neurons and presents bidirectional communication with the brain through afferent and efferent sensory fibers (Fishman and Dark \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Adipocyte size, lipid mobilization and paracrine secretion are controlled by WAT nerve endings (Bartness et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thus, sensory neurons that innervate the WAT are involved in the production of cytokines and the influx of immune cells, playing a central role in the low-grade inflammation observed in obesity (Bartness et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The increase vWAT in mice fed with HFD that we observed plays an important role in the development of mechanical hypersensitivity. Mechanical hyperalgesia can be triggered by the process of hypertrophy and hyperplasia of adipocytes in an inflammatory state. IL-1β is also a stimulus that massively activates BH4 synthesis via the \u003cem\u003ede novo\u003c/em\u003e pathway in immune cells, sensitizing nociceptive fibers and contributing to pain hypersensitivity (Cronin et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Eichwald et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fujita et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Latremoliere et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Scheffer and Latini 2020; Werner et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1990\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe relationship between BH4 and pain was first discovered, through the identification of an allele of the \u003cem\u003eGCH1\u003c/em\u003e gene haplotype that encodes for the GTP cyclohydrolase, the rate-limiting enzyme for BH4 biosynthesis, associated with reduced pain scores in multiple independent neuropathic pain cohorts (for review see Tegeder et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Based in this human validation of the biological role of BH4 in neuropathic and inflammatory pain, we demonstrated that excessive BH4 levels were produced by neurons in active pain, and immune cells infiltrating damaged nerves and inflamed tissues (Cronin et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fujita et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Latremoliere et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Based on this information, two inhibitors for BH4 production were developed (SPRi3 and QM385) and showed that inflammation and pain hypersensitivity were reduced along with decreased BH4 levels in targeted tissues (Cronin et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fujita et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Latremoliere et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, we discovered that the BH4-related metabolite, sepiapterin, accumulates in tissues and fluids exposed to the inhibitors (Fujita et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Urinary sepiapterin was also validated as a sensitive, specific, and non-invasive biomarker in a cohort of healthy humans receiving sulfasalazine, a pharmacological treatment approved by the FDA for treating inflammatory bowel diseases. Indeed, sulfasalazine was recently described to be an inhibitor of BH4 synthesis (Chidley et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These ground-breaking findings allowed us to hypothesize that excessive BH4 levels may play a significant role in pain development.\u003c/p\u003e \u003cp\u003eThe involvement of BH4 metabolism in animal models for inflammatory and neuropathic nociception showed a marked increase in BH4 synthesis in sensory neurons and nervous tissues inducing hyperalgesia (Latremoliere et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The data presented here showed that increased WAT accumulation also favored the synthesis of BH4, and possibly, the development of the observed mechanical and chemical hyperalgesia. Increased transcription of \u003cem\u003eGCH1\u003c/em\u003e and higher levels of BH4 have previously been reported in leukocytes infiltrating injured sciatic nerves, reinforcing the contribution of the immune system in the induction and maintenance of pain induced by the pathological production of BH4 (Latremoliere et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, the present data are in agreement with we our previous report showing increased levels of plasma neopterin, the by-product of BH4 metabolism that normally increases proportionally to BH4, in individuals affected by type III obesity (Lenoir da Silva et al. 2017).\u003c/p\u003e \u003cp\u003eBH4 is produced by macrophages that infiltrate tissues as a response to inflammation (Sakai, Kaufman, and Milstein \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) and its overproduction correlates with hyperalgesia (Latremoliere et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Thus, increased vWAT appears to be a favourable environment for the development of pain hypersensitivity. Indeed, the results demonstrated here showed concomitant increase in WAT, mechanical hyperalgesia, and BH4 levels. Furthermore, the urinary BH4 can be proposed as a potential easy-to-access, sensitive and reliable biomarker of pain development, and a promising target for the control of pain hypersensitivity in obesity. The use of pharmacological inhibitors aimed at reducing high levels of BH4 provide a potential therapeutic tool in the treatment of chronic pain.\u003c/p\u003e \u003cp\u003eThe literature has extensively demonstrated that the regular practice of moderate-intensity physical exercise is beneficial for health promotion, and for decreasing the risk of death from all causes (for a review see Bartness et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The Physical Activity Guidelines for Americans points out that in individuals affected by chronic diseases, the practice of moderate-intensity physical exercise for 150 min a week, or the practice of high-intensity aerobic physical activity for 75 min a week, are beneficial in improving the health of chronic illnesses affected individuals. Also, muscle-strengthening activities involving all major muscle groups, if practiced at least twice a week, provide additional health benefits (U.S. Department of Health and Human Services \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The effect of this intervention is more pronounced in the conditions in which the physiopathology is associated with persistent activation of the immune system (Nieman and Wentz 2019).\u003c/p\u003e \u003cp\u003eIt is known that the regular practice of moderate-intensity physical exercise reduces systemic inflammation. Our group demonstrated that lipopolysaccharide-induced inflammation in mice provoked increased glycemia levels in the GTT and led to an increase in the concentrations of urinary neopterin (Scheffer and Latini 2020), a sensitive marker of immune system activation (Huber et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). We also showed that the GTT and neopterin levels were normalised by physical exercise (Scheffer et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, we have also shown that neopterin is increased in the plasma of individuals affected by obesity type III (Lenoir da Silva et al. 2017), and insulin resistance-associated obesity, who also showed increased markers of inflammation (Remor et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, we have also shown that exercise modulated the inflammatory response induced by lipopolysaccharide. This anti-inflammatory status was characterized by reduced levels of neopterin in the urine (Scheffer et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Thus, regular practice of physical exercise positive modulates the anti-inflammatory response of the immune system normalizing the hypersensitivity scores seen, and possibly, the pathological production of BH4, in mice with increased vWAT.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThis study showed that increased vWAT elicited the overproduction and secretion of BH4 with consequent increased scores of chemical and mechanical hyperalgesias. Thus, urinary BH4 can be proposed as a biomarker of pain in obesity that is easy-to-access and no invasive. Finally, physical exercise can prevent the development of metabolic diseases and chronic pain, possibly by reducing WAT accumulation, BH4 overproduction, and therefore the associated inflammatory state.\u003c/p\u003e"},{"header":"6 Limitations of the study","content":"\u003cp\u003eUsing male mouse restricted our results since males and females often exhibit differences in metabolic composition due to the influence of sex hormones and various genetic and physiological factors. Male mice typically have a higher lean body mass, which can affect their metabolic rate, leading to variations in energy expenditure, nutrient utilization, and fat distribution.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e7 Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil; CNPq Universal #422488/2021-6).\u0026nbsp;A.L. is a CNPq fellow (CNPq PQ #312854/2019-6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8 Acknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the LAMEB/CCB-UFSC technicians for their assistance, and Ted Griswold for the English editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9 Conflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAL conceived the study with inputs from JMG and TE. AL and TE wrote the manuscript. TE, LB, AFS, DS, ACSP and JMG performed and designed the experimental strategy. AL and TE performed the statistical analysis and prepared all the figures. VM, ACS, CSS, RFS, MFR commented and edited the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAguiar, A. S., E. L. G. Moreira, A. A. Hoeller, P. A. Oliveira, F. M. C\u0026oacute;rdova, V. Glaser, R. Walz, R. A. Cunha, R. B. Leal, A. Latini, and R. D. S. Prediger. 2013. \u0026ldquo;Exercise Attenuates Levodopa-Induced Dyskinesia in 6-Hydroxydopamine-Lesioned Mice.\u0026rdquo; \u003cem\u003eNeuroscience\u003c/em\u003e 243:46\u0026ndash;53. doi: 10.1016/j.neuroscience.2013.03.039.\u003c/li\u003e\n\u003cli\u003eAguiar, Aderbal Silva, Marcelo Duzzioni, Aline Pertile Remor, Fabrine Sales Massafera Trist??o, Filipe C. 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Retrieved (https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight).\u003c/li\u003e\n\u003cli\u003eWoolf, Clifford J., and Qiufu Ma. 2007. \u0026ldquo;Nociceptors\u0026mdash;Noxious Stimulus Detectors.\u0026rdquo; \u003cem\u003eNeuron\u003c/em\u003e 55(3):353\u0026ndash;64. doi: 10.1016/j.neuron.2007.07.016.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"obesity, adipose tissue, sepiapterin reductase inhibitors, chronic pain, biomarker","lastPublishedDoi":"10.21203/rs.3.rs-4458806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4458806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eThis study aimed to examine the impact of high-fat diet (HFD)-induced obesity on pain sensitivity and tetrahydrobiopterin (BH4) levels. The effect of moderate-intensity physical exercise, an anti-inflammatory non-pharmacological intervention, on pain scores was also investigated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Adult male C57BL/J6 mice were fed standard or HFD for eight weeks. Total body weight, food intake, locomotor and motivational behavior and pain reflexes were measured. A subgroup of animals underwent physical exercise for five days per week over six weeks. Blood was collected for glucose tolerance testing and levels of lactate. Urine samples were collected to measure BH4 levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e We showed that HFD increased weight gain, visceral white adipose tissue, and the percentage of body fat. These anthropometric alterations were characterized by impaired glucose tolerance at four and eight weeks of the dietary intervention. It was also observed reduced locomotor activity and higher pain scores in HFD-fed mice that were prevented by the physical exercise intervention. HFD also induced the increase of urinary BH4 levels at four and eight weeks of intervention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Urinary BH4 can be proposed as a potential easy-to-access, sensitive and reliable biomarker of pain development, and a promising target for the control of pain hypersensitivity in obesity.\u003c/p\u003e","manuscriptTitle":"Pain hypersensitivity and increased urinary tetrahydrobiopterin levels in mice submitted to high fat diet","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-04 13:30:27","doi":"10.21203/rs.3.rs-4458806/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":"9d7f08dc-83eb-43b3-a60c-f3d2bbb5302b","owner":[],"postedDate":"June 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-05T19:55:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-04 13:30:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4458806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4458806","identity":"rs-4458806","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}