High dietary Fructose Drives Metabolic Dysfunction-Associated Steatotic Liver Disease via Activating ubiquitin-specific peptidase 2/11ß-hydroxysteroid dehydrogenase type 1 Pathway in Mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver-related morbidity and mortality. Though high fructose intake is acknowledged as a metabolic hazard, its role in the etiology of MASLD requires further clarification. Here, we demonstrated that high dietary fructose drives MASLD development and promotes MASLD progression in mice, and identified Usp2 as a fructose-responsive gene in the liver. Elevated USP2 levels were detected in the hepatocytes of MASLD mice; a similar increase was observed following fructose exposure in primary hepatocytes and mouse AML12 cells. Notably, hepatocytes overexpressing USP2 presented with exaggerated lipid accumulation and metabolic inflammation when exposed to fructose. Conversely, USP2 knockdown mitigated these fructose-induced changes. Furthermore, USP2 was found to activate the C/EBPα/11ß-HSD1 signaling, which further impacted the equilibrium of cortisol and cortisone in the circulation of mice. Collectively, our findings revealed the role of dietary fructose in MASLD pathogenesis and identified the USP2-mediated C/EBPα/ 11ß-HSD1 signaling as a potential target for the management of MASLD.

Ileum Proteomics Identifies Distinct Pathways Associated with Different Dietary Doses of Copper-Fructose Interactions: Implications for the Gut-Liver Axis and MASLD.

The interactions of different dietary doses of copper with fructose contribute to the development of metabolic dysfunction-associated steatotic liver disease (MASLD) via the gut-liver axis. The underlying mechanisms remain elusive. The aim of this study was to identify the specific pathways leading to gut barrier dysfunction in the ileum using a proteomics approach in a rat model. Male weanling Sprague Dawley rats were fed diets with adequate copper (CuA), marginal copper (CuM), or supplemented copper (CuS) in the absence or presence of fructose supplementation (CuAF, CuMF, and CuSF) for 4 weeks. Ileum protein was extracted and analyzed with an LC-MS. A total of 2847 differentially expressed proteins (DEPs) were identified and submitted to functional enrichment analysis. As a result, the ileum proteome and signaling pathways that were differentially altered were revealed. Of note, the CuAF is characterized by the enrichment of oxidative phosphorylation and ribosome as analyzed with the KEGG; the CuMF is characterized by an enriched arachidonic acid metabolism pathway; and focal adhesion, the regulation of the actin cytoskeleton, and tight junction were significantly enriched by the CuSF. In conclusion, our proteomics analysis identified the specific pathways in the ileum related to the different dietary doses of copper-fructose interactions, suggesting that distinct mechanisms in the gut are involved in the development of MASLD.

Effects of Liquid Fructose Supplementation and Chronic Unpredictable Stress on Uterine Contractile Activity in Nonpregnant Rats.

Increased fructose consumption and chronic stress, the major characteristics of modern lifestyle, impact human health; however, the consequences of their combination on the uterus remain understudied. In this study, we investigated contractile activity, morphology, and intracellular activity of antioxidant enzymes in uteri from virgin Wistar rats subjected to liquid fructose supplementation and/or unpredictable stress over 9 weeks. Contractile activity and uterine response to oxytocin or adrenaline were examined ex vivo using isolated bath chambers. Fructose supplementation, irrespective of stress, affected uterine morphology by increasing endometrium while decreasing myometrium volume density, attenuated uterine response to increasing doses of oxytocin, and increased glutathione peroxidase activity. Stress, irrespective of fructose, attenuated dose-dependent adrenaline-induced uterine relaxation. Stress, when applied solely, decreased mitochondrial superoxide dismutase activity. In the combined treatment, irregular estrous cycles and both reduced response to oxytocin and to adrenaline (as a consequence of fructose consumption and exposure to stress), along with fructose-related alteration of uterine morphology, were detected. In conclusion, fructose and stress affect uterine contractile activity, irrespective of each other, by inducing completely distinct responses in isolated uteri. In the combined treatment, the effects of both factors were evident, suggesting that the combination exerts more detrimental effects on the uterus than each factor individually.

Magnoflorine Ameliorates Chronic Kidney Disease in High-Fat and High-Fructose-Fed Mice by Promoting Parkin/PINK1-Dependent Mitophagy to Inhibit NLRP3/Caspase-1-Mediated Pyroptosis.

Excessive intake of fat and fructose in Western diets has been confirmed to induce renal lipotoxicity, thereby driving the progression of chronic kidney disease (CKD). This study was conducted to evaluate the efficacy of magnoflorine in a CKD mouse model subjected to high-fat and high-fructose diets. Our results demonstrated that magnoflorine treatment ameliorated abnormal renal function indices (serum creatinine, urea nitrogen, uric acid, and urine protein) in high-fat- and high-fructose-fed mice. Histologically, renal tubular cell steatosis, lipid deposition, tubular dilatation, and glomerular fibrosis were significantly reduced by the magnoflorine treatment in these mice. Mechanistically, magnoflorine promotes Parkin/PINK1-mediated mitophagy, thereby inhibiting NLRP3/Caspase-1-mediated pyroptosis. Consistent findings were observed in the palmitic acid-incubated HK-2 cell model. Notably, both silencing of Parkin and the use of a mitophagy inhibitor reversed the inhibitory effect of magnoflorine on NLRP3 inflammasome activation in vitro. Therefore, the present study provides compelling evidence that magnoflorine improves renal injury in high-fat- and high-fructose-fed mice by promoting Parkin/PINK1-dependent mitophagy to inhibit NLRP3 inflammasome activation and pyroptosis. Our findings suggest that dietary supplementation with magnoflorine and magnoflorine-rich foods (such as magnolia) might be an effective strategy for the prevention of CKD.

Peripheral inflammatory hyperalgesia is exacerbated in rats with metabolic disorders induced by a fructose diet.

This study explored the effects of fructose-induced obesity and metabolic disorders on peripheral inflammatory hyperalgesia, employing quantitative sensory testing with the von Frey test and measuring paw edema to assess inflammatory responses. Wistar rats were administered water or 10% fructose solution ad libitum over a period of 5 weeks. After intraplantar administration of inflammatory agents such as carrageenan (1 mg/paw), lipopolysaccharide (LPS; 100 µg/paw), or prostaglandin E2 (PGE2, 100 ng/paw), we conducted mechanical hyperalgesia tests and paw edema evaluations. The fructose diet resulted in dyslipidemia, elevated insulin and leptin plasma levels, insulin resistance, and increased epididymal and retroperitoneal adiposity compared to control animals. In response to inflammatory agents, the fructose group displayed significantly enhanced peripheral hyperalgesia and more pronounced paw edema. Our results demonstrate that fructose not only contributes to the development of obesity and metabolic disorder but also exacerbates peripheral inflammatory pain responses by enhancing prostaglandin sensitivity.

Modulatory effects of rutin and vitamin A on hyperglycemia induced glycation, oxidative stress and inflammation in high-fat-fructose diet animal model.

In the current study we investigated the impact of combination of rutin and vitamin A on glycated products, the glyoxalase system, oxidative markers, and inflammation in animals fed a high-fat high-fructose (HFFD) diet. Thirty rats were randomly divided into six groups (n = 5). The treatments, metformin (120 mg/kg), rutin (100 mg/kg), vitamin A (43 IU/kg), and a combination of rutin (100 mg/kg) and vitamin A (43 IU/kg) were given to relevant groups of rats along with high-fructose high-fat diet for 42 days. HbA1c, D-lactate, Glyoxylase-1, Hexokinase 2, malondialdehyde (MDA), glutathione peroxidase (GPx), catalase (CAT), nuclear transcription factor-B (NF-κB), interleukin-6 (IL-6), interleukin-8 (IL-8) and histological examinations were performed after 42 days. The docking simulations were conducted using Auto Dock package. The combined effects of rutin and vitamin A in treated rats significantly (p < 0.001) reduced HbA1c, hexokinase 2, and D-lactate levels while preventing cellular damage. The combination dramatically (p < 0.001) decreased MDA, CAT, and GPx in treated rats and decreased the expression of inflammatory cytokines such as IL-6 andIL-8, as well as the transcription factor NF-κB. The molecular docking investigations revealed that rutin had a strong affinity for several important biomolecules, including as NF-κB, Catalase, MDA, IL-6, hexokinase 2, and GPx. The results propose beneficial impact of rutin and vitamin A as a convincing treatment strategy to treat AGE-related disorders, such as diabetes, autism, alzheimer's, atherosclerosis.

Fructose-induced metabolic reprogramming of cancer cells.

Excess dietary fructose consumption has been long proposed as a culprit for the world-wide increase of incidence in metabolic disorders and cancer within the past decades. Understanding that cancer cells can gradually accumulate metabolic mutations in the tumor microenvironment, where glucose is often depleted, this raises the possibility that fructose can be utilized by cancer cells as an alternative source of carbon. Indeed, recent research has increasingly identified various mechanisms that show how cancer cells can metabolize fructose to support their proliferating and migrating needs. In light of this growing interest, this review will summarize the recent advances in understanding how fructose can metabolically reprogram different types of cancer cells, as well as how these metabolic adaptations can positively support cancer cells development and malignancy.

Maternal fructose intake aggravates the harmful effects of a Western diet in rat male descendants impacting their cholesterol metabolism.

Scope: fructose consumption from added sugars correlates with the epidemic rise in MetS and CVD. Maternal fructose intake has been described to program metabolic diseases in progeny. However, consumption of fructose-containing beverages is allowed during gestation. Cholesterol is also a well-known risk factor for CVD. Therefore, it is essential to study Western diets which combine fructose and cholesterol and how maternal fructose can influence the response of progeny to these diets. Methods and results: a high-cholesterol (2%) diet combined with liquid fructose (10%), as a model of an unhealthy Western diet, was administered to descendants from control and fructose-fed mothers. Gene (mRNA and protein) expression and plasma, fecal and tissue parameters of cholesterol metabolism were measured. Interestingly, progeny from fructose-fed dams consumed less liquid fructose and cholesterol-rich chow than males from control mothers. Moreover, descendants of fructose-fed mothers fed a Western diet showed an increased cholesterol elimination through bile and feces than males from control mothers. Despite these mitigating circumstances to develop a proatherogenic profile, the same degree of hypercholesterolemia and severity of steatosis were observed in all descendants fed a Western diet, independently of maternal intake. An increased intestinal absorption of cholesterol, synthesis, esterification, and assembly into lipoprotein found in males from fructose-fed dams consuming a Western diet could be the cause. Moreover, an augmented GLP2 signalling seen in these animals would explain this enhanced lipid absorption. Conclusions: maternal fructose intake, through a fetal programming, makes a Western diet considerably more harmful in their descendants than in the offspring from control mothers.

Fennel seeds extract prevents fructose-induced cardiac dysfunction in a rat model of metabolic syndrome via targeting abdominal obesity, hyperuricemia and NF-κß inflammatory pathway.

BACKGROUND: Metabolic syndrome (MetS) is commonly associated with increased risk of cardiac disease that affects a large number of world populations. OBJECTIVE: This research attempted to investigate the efficacy of fennel seeds extract (FSE) in preventing development of cardiac dysfunction in rats on fructose enriched diet for 3 months, as a model of MetS. MATERIALS & METHODS: Thirty adult Wistar male rats (160-170 g) were assigned into 5 groups including control, vehicle, FSE (200 mg/kg BW) and fructose (60%) fed rats with and without FSE. Following the last treatment, blood pressure, ECG and heart rate were measured. Next, blood and cardiac tissues were taken for biochemical and histological investigations. RESULTS: Feeding fructose exhibited characteristic features of MetS involving, hypertension, abnormal ECG, elevated heart rate, serum glucose, insulin, lipids and insulin resistance, accompanied by abdominal obesity, cardiac hypertrophy and hyperuricemia. Fructose fed rats also showed significant reduction in cardiac antioxidants (GSH, SOD, CAT) with elevation in oxidative stress indices (NADPH oxidase, O2.-, H2O2, MDA, PCO), NF-κß, pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), adhesion molecules (ICAM-1, VCAM-1) and serum cardiac biomarkers (AST, LDH, CK-MB, cTn-I). Histopathological changes evidenced by destruction of cardiac myofibrils, cytoplasmic vacuolization, and aggregation of inflammatory cells were also detected. Consumption of FSE showed high ability to alleviate fructose-induced hypertension, ECG abnormalities, cardiac hypertrophy, metabolic alterations, oxidative stress, inflammation and histological injury. CONCLUSION: Findings could suggest FSE as a complementary supplement for preventing MetS and associated cardiac outcomes. However, well controlled clinical studies are still needed.

Mechanisms Underlying Memory Impairment Induced by Fructose.

Fructose consumption has increased over the years, especially in adolescents living in urban areas. Growing evidence indicates that daily fructose consumption leads to some pathological conditions, including memory impairment. This review summarizes relevant data describing cognitive deficits after fructose intake and analyzes the underlying neurobiological mechanisms. Preclinical experiments show sex-related deficits in spatial memory; that is, while males exhibit significant imbalances in spatial processing, females seem unaffected by dietary supplementation with fructose. Recognition memory has also been evaluated; however, only female rodents show a significant decline in the novel object recognition test performance. According to mechanistic evidence, fructose intake induces neuroinflammation, mitochondrial dysfunction, and oxidative stress in the short term. Subsequently, these mechanisms can trigger other long-term effects, such as inhibition of neurogenesis, downregulation of trophic factors and receptors, weakening of synaptic plasticity, and long-term potentiation decay. Integrating all these neurobiological mechanisms will help us understand the cellular and molecular processes that trigger the memory impairment induced by fructose.

A cross talk study on sitagliptin mediated reclamation on TGF ß signalling, DPP 4, miR-29a and miR-24 expression in PCOS rats fed with high fat-high fructose diet.

Polycystic Ovary Syndrome (PCOS) is a multifactorial reproductive, endocrine and metabolic disturbance which is very commonly observed in females of reproductive age group. The disease is still incurable however the use of synthetic drugs in combination with lifestyle is recommended. Accordingly, the present study was conducted to investigate the possible beneficial effects of sitagliptin on PCOS induced rats on control diet (CD)/high fat- high fructose diet (HFFD). PCOS was induced by giving testosterone propionate (TP) for 28 days to both the CD/HFFD rats and treated with STG i.p. for last 15 days. At the end of the experiment lipid profile, inflammatory markers, expression of NF-κB-p65, miR-24 and miR-29a, fibrotic and apoptotic proteins from ovary tissue were examined. Moreover, lipid accumulation and fibrosis of ovary tissue was further confirmed using Sudan III and Masson's trichrome stain. STG treated rats exerted a significant decrease in levels of cholesterol, TG, LDL-C, VLDL-C, IL-6 and TNF-α and increased HDL-C level, miR-24 and miR-29a expression. STG treated groups expressed significantly decreased expression of NF-κB-p65, TGF-ß1, p-Smad 2 and p-Smad 3 followed by no significant changes in the expression of BAX, caspase-9, caspase-3 and Bcl-2 in all the PCOS induced groups. Among all the CD/ HFFD fed groups, rats on HFFD showed more devastating effect which suggests that diet plays a major role in genesis of PCOS. In conclusion, current results reflect the potential impact of STG against dyslipidaemia, inflammation and fibrosis in PCOS rats via regulating dyslipidaemia and fibrosis via DPP 4 mediated miR-29a expression.

Dietary Fructose and Sodium Consumed during Early Mid-Life Are Associated with Hypertensive End-Organ Damage by Late Mid-Life in the CARDIA Cohort.

We aimed to investigate how dietary fructose and sodium impact blood pressure and risk of hypertensive target organ damage 10 years later. Data from n = 3116 individuals were obtained from the Coronary Artery Risk Development in Young Adults (CARDIA) study. Four groups were identified based on the four possible combinations of the lower and upper 50th percentile for sodium (in mg) and fructose (expressed as percent of total daily calories). Differences among groups were ascertained and logistic regression analyses were used to assess the risk of hypertensive target organ damage (diastolic dysfunction, coronary calcification and albuminuria). Individuals in the low-fructose + low-sodium group were found to have lower SBP compared to those in the low-fructose + high-sodium and high-fructose + high-sodium groups (p < 0.05). The highest risk for hypertensive target organ damage was found for albuminuria only in the high-fructose + high-sodium group (OR = 3.328, p = 0.006) while female sex was protective across all groups against coronary calcification. Our findings highlight that sodium alone may not be the culprit for hypertension and hypertensive target organ damage, but rather when combined with an increased intake of dietary fructose, especially in middle-aged individuals.

The Impact of Excessive Fructose Intake on Adipose Tissue and the Development of Childhood Obesity.

Worldwide, childhood obesity cases continue to rise, and its prevalence is known to increase the risk of non-communicable diseases typically found in adults, such as cardiovascular disease and type 2 diabetes mellitus. Thus, comprehending its multiple causes to build healthier approaches and revert this scenario is urgent. Obesity development is strongly associated with high fructose intake since the excessive consumption of this highly lipogenic sugar leads to white fat accumulation and causes white adipose tissue (WAT) inflammation, oxidative stress, and dysregulated adipokine release. Unfortunately, the global consumption of fructose has increased dramatically in recent years, which is associated with the fact that fructose is not always evident to consumers, as it is commonly added as a sweetener in food and sugar-sweetened beverages (SSB). Therefore, here, we discuss the impact of excessive fructose intake on adipose tissue biology, its contribution to childhood obesity, and current strategies for reducing high fructose and/or free sugar intake. To achieve such reductions, we conclude that it is important that the population has access to reliable information about food ingredients via food labels. Consumers also need scientific education to understand potential health risks to themselves and their children.

Effect of a high-fat high-fructose diet on the composition of the intestinal microbiota and its association with metabolic and anthropometric parameters in a letrozole-induced mouse model of polycystic ovary syndrome.

OBJECTIVE: It has been suggested that dysbiosis of the gut microbiota is associated with the pathogenesis of Polycystic Ovary Syndrome (PCOS), and that improper diet can aggravate these changes. This study thus aimed to investigate the effects of a high-fat/high-fructose (HF/HFr) diet on the gut microbial community and their metabolites in prepubertal female mice with letrozole (LET)-induced PCOS. We also tested the correlations between the relative abundance of microbial taxa and selected PCOS parameters. RESEARCH METHODS & PROCEDURES: Thirty-two C57BL/6 mice were randomly divided into four groups (n = 8) and implanted with LET or a placebo, with simultaneous administration of a HF/HFr diet or standard diet (StD) for 5 wk. The blood and intestinal contents were collected after the sacrifice. RESULTS: Placebo + HF/HFr and LET + HF/HFr had significantly higher microbial alpha diversity than either group fed StD. The LET-implanted mice fed StD had a significantly higher abundance of Prevotellaceae_UCG-001 than the placebo mice fed StD. Both groups fed the HF/HFr diet had significantly lower fecal levels of short-chain fatty acids than the placebo mice fed StD, while the LET + HF/HFr animals had significantly higher concentrations of lipopolysaccharides in blood serum than either the placebo or LET mice fed StD. Opposite correlations were observed between Turicibacter and Lactobacillus and the lipid profile, CONCLUSION: HF/HFr diet had a much stronger effect on the composition of the intestinal microbiota of prepubertal mice than LET itself.

Whey protein supplementation reduced the liver damage scores of rats fed with a high fat-high fructose diet.

Different functional foods with bioactive nutrients are being explored for the management of NAFLD. Whey proteins are rich in bioactive peptides and are suggested to show antioxidant and anti-inflammatory effects. We aim to test the hypothesis that the whey protein supplementation following a high fat-high fructose (HFHF) diet would protect against liver damage, inflammation, endotoxemia and steatosis in male Wistar rats. 36 rats were randomized into four groups for 8 weeks as the HFHF diet group, HFHF diet and whey protein isolate (WPI-200mg/kg/day) group (HFHF+WPI), control (C) group, and C+WPI (200mg/kg/day) group. Rats fed with a HFHF diet had higher final body weight compared to C and C+WPI groups (p = 0.002). Thus, WPI showed no significant effects for the body weight of rats with a HFHF diet. On the other hand, the HFHF+WPI group had significantly lower abdominal circumference when compared with the HFHF group (p<0,001). Higher serum CRP levels were observed in the groups with a HFHF diet (p<0,001) and WPI supplementation showed no effects on CRP levels. Whey protein supplementation resulted with lower total liver damage score in HFHF+WPI group compared with the HFHF diet group (p<0,001). Conversely, higher liver damage scores were observed with the C+WPI group compared to C group (p<0,001). HFHF diet resulted with higher expression of TLR-4 in the liver meanwhile WPI supplementation showed no effects on liver TLR-4 expression. We observed higher colon Occludin expression in HFHF+WPI and C+WPI groups compared with HFHF and C groups (p<0,001). Our results showed that, whey protein supplementation might help improve liver damage associated with a high fat-high fructose diet and increase the expression of Occludin in the small intestine and colon.

A short-term rodent model for non-alcoholic steatohepatitis induced by a high-fat diet and carbon tetrachloride.

Several models of mice-fed high-fat diets have been used to trigger non-alcoholic steatohepatitis and some chemical substances, such as carbon tetrachloride. The present study aimed to evaluate the joint action of a high-fat diet and CCl4 in developing a short-term non-alcoholic steatohepatitis model. C57BL6/J mice were divided into two groups: standard diet-fed (SD), the high-fat diet-fed (HFD) and HFD + fructose-fed and carbon tetrachloride (HFD+CCl4). The animals fed with HFD+CCl4 presented increased lipid deposition compared with both SD and HFD mice. Plasma cholesterol was increased in animals from the HFD+CCl4 group compared with the SD and HFD groups, without significant differences between the SD and HFD groups. Plasma triglycerides showed no significant difference between the groups. The HFD+CCl4 animals had increased collagen deposition in the liver compared with both SD and HFD groups. Hydroxyproline was also increased in the HFD+CCl4 group. Liver enzymes, alanine aminotransferase and aspartate aminotransferase, were increased in the HFD+CCl4 group, compared with SD and HFD groups. Also, CCl4 was able to trigger an inflammatory process in the liver of HFD-fed animals by promoting an increase of ∼2 times in macrophage activity, ∼6 times in F4/80 gene expression, and pro-inflammatory cytokines (IL-1b and TNFa), in addition to an increase in inflammatory pathway protein phosphorylation (IKKbp). HFD e HFD+CCl4 animals increased glucose intolerance compared with SD mice, associated with reduced insulin-stimulated AKT activity in the liver. Therefore, our study has shown that short-term HFD feeding associated with fructose and CCl4 can trigger non-alcoholic steatohepatitis and cause damage to glucose metabolism.

Fructose induces hepatic steatosis in adolescent mice linked to the disorders of lipid metabolism, bile acid metabolism, and autophagy.

The effects of excessive fructose intake on the development and progression of metabolic disorders have received widespread attention. However, the deleterious effects of fructose on the development of hepatic metabolic disease in adolescents and its potential mechanisms are not fully understood. In this study, we investigated the effects of isocaloric fructose-rich diets on the liver of adolescent mice. The results showed that fructose-rich diets had no effect on the development of obesity in the adolescent mice, but did induce hepatic lipid accumulation. Besides, we found that fructose-rich diets promoted hepatic inflammatory responses and oxidative stress in adolescent mice, which may be associated with activation of the NLRP3 inflammasome and inhibition of the Nrf2 pathway. Furthermore, our results showed that fructose-rich diets caused disturbances in hepatic lipid metabolism and bile acid metabolism, as well as endoplasmic reticulum stress and autophagy dysfunction. Finally, we found that the intestinal barrier function was impaired in the mice fed fructose-rich diets. In conclusion, our study demonstrates that dietary high fructose induces hepatic metabolic disorders in adolescent mice. These findings provide a theoretical foundation for fully understanding the effects of high fructose intake on the development of hepatic metabolic diseases during adolescence.

Caffeine Decreases Topiramate Levels in Zebrafish Larvae in a Pentylenetetrazol-Induced Seizure Model.

Epilepsy ranks as the second-most prevalent neurological disease, and is characterized by seizures resulting in neurobiological and behavioral impairment. Naturally occurring in coffee beans or tea leaves, the alkaloid caffeine (CAF) is the most prevalent global stimulant. Caffeine has been observed to influence epileptic seizures and the efficacy of antiepileptic medications, with a notable impact on topiramate (TPM). This study aimed to explore the influence of CAF on TPM's anticonvulsant effects in zebrafish larvae within a PTZ-induced seizure model, concurrently determining TPM concentrations through a sophisticated analytical approach based on ultrahigh-performance liquid chromatography and subsequent mass spectrometric detection. Zebrafish larvae four days post-fertilization were incubated for 18 h with varying doses of TPM or combinations of CAF + TPM, and locomotor activity was then assessed. Seizures were induced by introducing a PTZ solution to achieve a final concentration of 20 mM. Utilizing liquid chromatography-mass spectrometry (LC-MS/MS), TPM levels in the larvae were quantified. CAF co-administration (especially in higher doses) with TPM caused a decrease in the average locomotor activity in the larvae compared to TPM alone. Moreover, CAF decreased TPM levels in the larvae at all investigated doses. In conclusion, these findings offer a novel perspective on the interplay between CAF and TPM, shedding light on previously unexplored facets. The potential impact of CAF consumption in assisting with epileptic seizure control, unless proven otherwise, suggests a noteworthy consideration for future research and clinical practices.