Effects of kaempherol-3-rhamnoside on metabolic enzymes and AMPK in the Liver Tissue of STZ-Induced Diabetes in Mice

Effects of kaempherol-3-rhamnoside on metabolic enzymes and AMPK in the Liver Tissue of STZ-Induced Diabetes in Mice | 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 Effects of kaempherol-3-rhamnoside on metabolic enzymes and AMPK in the Liver Tissue of STZ-Induced Diabetes in Mice Alhussain H. Aodah, Faisal K Alkholifi, Sushma Devi, Ahmed I. Foudah, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3930074/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 The current study aimed to investigate the potential antidiabetic properties of kaempherol-3-rhamnoside (Afzelin), both alone and in combination with insulin. To accomplish this, different groups of animals received kaempherol-3-rhamnoside doses and combinations of Humalog insulin and kaempherol-3-rhamnoside for 28 days. The objective was to evaluate the role of kaempherol-3-rhamnoside in glycolytic, gluconeogenic and NADP-linked lipogenic enzymes in liver tissues from STZ-induced diabetic mice while examining pharmacological modulations within the AMPK pathway. These could further regulate metabolic enzymes. The results indicated that in diabetic mice, glycolytic enzyme activities were significantly lower while gluconeogenic ones were higher; however, lipid-based enzyme activity decreased. It was observed that kaempherol-3-rhamnoside had a therapeutic role in the treatment of diabetes by normalising enzyme activities involved in glucose and lipid metabolism. Furthermore, kaempherol-3-rhamnoside treatment activated AMPK activity within liver tissues in diabetic mice by increasing the p-AMPK/AMPK ratio. The inhibited AMPK activity observed in these mice was overcome with this treatment. Additionally, the biochemical analysis indicated that kaempherol-3-rhamnoside has the potential to restore cellular function at the molecular level. kaempherol-3-rhamnoside diabetes glycolytic enzymes AMPK insulin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Diabetes mellitus (DM) refers to a cluster of metabolic disorders that cause elevated levels of glucose, lipids, amino acids, and lactate circulating in the body. The root causes of DM include inadequate insulin secretion from the pancreas or diminished sensitivity to insulin or both simultaneously. It affects both insulin secretion and action resulting in hyperinsulinemia [ 1 , 2 ]. According to the World Health Organisation, it is predicted that by 2030, there will be more than twice as many people worldwide who have DM compared to the numbers recorded in 2000. The estimated figure could surpass a staggering total of 300 million people affected by this condition [ 3 ]. To effectively manage diabetes, the ultimate aim is to regulate blood sugar levels in both type 1 and type 2 cases. Achieving this requires efficient glycolysis, which transforms glucose into energy that manages insulin and cell activity. Enzymes such as glucokinase and 6-phosphofructo-1-kinase control the glycolysis rate. Enzymes vary in their regulation between cell types. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme is also crucial in this process [ 4 ]. Enzymes respond to signals from nutrition and hormones, which affect their transcription, translation, and post-translational modifications. The intricate process of hepatic glucose production is essentially a result of the complex interplay between enzymes and metabolic pathways within hepatocytes, which work together to regulate glycolysis [ 4 ]. When there is too much, the latter can lead to high blood sugar levels in people with diabetes. Glycolysis results in glucose-stimulated insulin release in pancreatic beta cells. Hyperglycemia can occur when there is a deficiency of circulating insulin or when its function is impaired [ 5 ]. Adipocytes use glycolysis to produce metabolites that facilitate lipogenesis and redirect surplus fatty acids toward the synthesis of triglycerides. This mechanism curtails oxidative stress within cells. Adipocytes generate pro-hyperglycemic factors when the body is in a state of increased inflammation, leading to insulin resistance and elevated blood glucose levels [ 6 ]. When insulin levels are inadequate or the body becomes resistant to it, there is a malfunctioning of glycolysis. This is caused by the metabolic and regulatory enzymes involved in glycolysis functioning inappropriately in terms of quantity and/or activity. Effective strategies to manage diabetes and its associated complications may include measures to improve glycolytic activity by modulating key metabolic and regulatory enzymes [ 7 ] [ 8 ]. The association of enzymes affects glucose oxidation. The high association of enzymes favours glucose for fermentation and biosynthesis. In skeletal and cardiac muscles of diabetic patients, glycolysis undergoes down-regulation due to hexokinase and phosphofructokinase enzyme deficiency. Cancer conditions commonly show elevated glycolytic rates associated with this phenomenon [ 9 ]. The appropriate utilisation of glucose by various tissues in the body, including the liver, pancreas, and kidneys, is critical in diabetes to prevent the accumulation and toxicity of cellular glucose of cellular glucose that can lead to serious complications. This highlights the importance of regulating both under- and over-use of glucose. Insulin-dependent diabetes mellitus is characterised by metabolic dysfunction primarily resulting from hyperinsulinemia and hyperglycemia. The maintenance of carbohydrate and lipid homeostasis depends on the equilibrium between their production and usage in the main peripheral tissues, which is substantially altered in individuals with diabetes [ 10 , 11 , 12 ]. This mechanism is key to developing new antidiabetic drugs. The 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway effectively regulates diabetes. Activation of the AMPK protein can increase glucose uptake and suppress intracellular glucose production. This promotes optimal cell function, as shown in several studies. Impaired AMPK activity is common among people with diabetes, making activation of this protein a persuasive solution. In particular, drugs used to treat this condition, including metformin, exert their therapeutic effects by regulating AMPK function. As a result, pharmacological substances with promising potential as therapeutic choices for the treatment of DM include those that can efficiently activate and modulate AMPK activity [ 13 , 14 ]. It is important to consider alternative treatments based on scientific research for patients who experience adverse effects from allopathic drugs, including hypoglycemia. Plant-based medications such as berberine, quercetin, and resveratrol can effectively regulate the AMPK pathway to manage diabetes mellitus without harmful side effects [ 15 ]. Insulin therapy and oral drugs such sulfonylureas, biguanides, -glucosidase inhibitors, and glinides are often used to manage diabetes. However, in resource-limited settings where access to these drugs is limited due to high costs and unavailability; adopting alternative therapies or preventive measures becomes crucial. To provide optimal diabetes care, healthcare providers should explore lifestyle changes and natural remedies [ 16 ]. In the world of healthcare, there is a new wave of interest in herbal remedies. This can be attributed to the harmful side effects associated with taking oral hypoglycemic agents, which are often prescribed to treat diabetes mellitus. With more and more people looking for natural alternatives to traditional medications, it is crucial that we embrace this shift towards holistic healing methods. Herbal remedies have been used for centuries to treat various ailments and now offer a safe solution for those seeking relief from their diabetic symptoms without risking dangerous side effects. Traditional plant-based remedies are vital in treating DM. Based on scientific evidence, these traditional remedies have proven effective in controlling blood sugar levels and alleviating the harmful effects of this persistent medical condition [ 17 ]. Kaempherol-3-rhamnoside is a flavonol glycoside that naturally occurs in plants. It is derived primarily from the sources of Ficus palmata and Nymphaea odoratam . kaempherol-3-rhamnoside belongs to the class of phytochemicals known as flavonoids, which are widely recognised for their medicinal properties and health benefits [ 18 ]. kaempherol-3-rhamnoside is an exceptional natural compound that acts as both a potent inhibitor of NOS and NADPH oxidase. Its impressive therapeutic potential is attributed to its remarkable multifunctional properties, which include powerful antibacterial, anti-inflammatory, antiapoptotic, and antitumour activities. It is no wonder why kaempherol-3-rhamnoside continues to be a promising candidate for future drug development efforts. kaempherol-3-rhamnoside might serve as an effective treatment against P. aeruginosa -related diseases [ 19 ]. While promising results have been observed in animal studies, further research is needed to determine the efficacy and safety of kaempherol-3-rhamnoside as a potential treatment for breast [ 20 ]. We investigated the effects of kaempherol-3-rhamnoside on metabolic and oxidative enzymes in diabetic mice. This study is significant to understanding the benefits of using kaempherol-3-rhamnoside to manage diabetes, which could lead to more effective treatments. Additionally, we found pharmacological modulation of the AMPK pathway as shown in Fig. 1 . 2. Materials and Methods Chemicals Streptozotocin purchased from Sisco Research Laboratories Pvt. Ltd., India. Glu-cose kit (120200- Erba Mannheim, India), Insulin ELISA Kit (Millipore, Billerica, MA, USA) were used. Cholesterol, triglycerides, high-density lipoproteins and free fatty ac-ids were assessed using assay kits purchased from Spin react, Spain. Anti-AMPK and anti-p-AMPK from Abcam Inc., Cambridge, MA. The enzyme kits were purchased from: hexokinase type I enzyme - ab136957, Colorimetric assay kit from Abcam Inc.); phosphofructokinase- MAK093, and pyruvate kinase - MAK072 from Sigma Aldrich, India; lactate dehydrogenase, glucose-6-phosphatase and fructose-1,6-bisphosphatase from BT LAB, Bioassay Technology Laboratory, India. It is imperative that all reagents administered to animals must adhere to pharmacological and chemical grade stand-ards, ensuring they are free from contamination. Acute Oral Toxicity Study A study was conducted to evaluate the acute oral toxicity of the substance in ac-cordance with guidelines provided by the OECD (Organisation for Economic Coopera-tion and Development) No. 423. In this study, albino mice with weight ranging from 18 to 28 g and male gender were used. To ensure consistency in drug administration and minimise variability between individuals, all mice underwent an overnight fast prior to receiving any drugs or treatments. Three mice received a robust single oral dose equivalent to 500 mg/kg of their body weight, courtesy of the powerful compound known as kaempherol-3-rhamnoside. After giving kaempherol-3-rhamnoside, food was not allowed for the next 4–5 hours. The animals were carefully monitored as indi-viduals within the first half hour of being dosed and then at various intervals throughout the initial 24 hours (with a particular focus on the first four). Following this initial period, they were observed daily for two weeks. Overall, their well-being was closely monitored over an extended period of time. Daily, observations were made on the animal's appearance and behaviour including skin, fur, eyes, nose, breathing rate, heart rate, blood pressure, and other bodily functions such as saliva production, tear secretion, sweating, piloerection (hair standing), involuntary urination or defeca-tion along with changes in drowsiness, gait, tremors, and seizures. Any deaths that occurred within 14 days were recorded [ 21 ]. Animals Male albino mice, weighing 18-28g and between 7–8 weeks old, were kept in a temperature-controlled room with a 12-hour light-dark cycle. They were provided ad libitum access to the local market of Saudi Arabia, as well as tap water for drinking. The food cups were replenished daily. This protocol was approved by the Internal Animal Ethics Committee with authorisation number SCBR-024-2022. The complete experimental work flow is shown in flow diagram 1. Flow diagram 1: Experimental design Experimental design Induction of Diabetes The development of DM was begun by administering a single ip dose of 65 mg/kg STZ that had been dissolved in citrate buffer (100 mM, pH 4.5) according to the meth-od described in the reference source provided [ 22 ]. All animals involved in the ex-periment were fed a high-fat diet, except the normal control group, who received cit-rate buffer only. After four days, mice that showed hyperglycemia (measured at ≥ 250 mg/dL) were classified as diabetic. Categorised mice with diabetes into different groups, Group I normal control (only vehicle) and Group II diabetic control (only STZ given). Group III diabetic mice were treated with an injection of regular human insulin (Humalog 2 U/kg) for 28 days. Group IV kaempherol-3-rhamnoside (2.5 mg/kg), Group V (5 mg/kg), and Group VI (human insulin 1U/kg and kaempherol-3-rhamnoside 2.5 mg/kg). The mice received treatment for a period of 28 days, after which they were fasted and weighed. Additionally, blood samples were collected from the retroorbital area. The mice were then anaesthetised and then euthanised by cervical decapitation. Subsequently, the pancreas and liver tissue were isolated, weighed and stored in a deep freezer with a temperature of -70°C [ 23 – 24 ]. Fasting blood glucose level The fasting blood glucose level was recorded on days 0, 14, 21 and 28. Blood sam-ples were taken from the retro orbital area and the findings were reported as milli-grams per deciliter (mg/dl) [ 25 ]. Blood glucose levels were measured using a glucome-ter (accu-check). Plasma insulin level On the 28th day, plasma insulin levels were measured using a Rat/Mouse Insulin ELISA Kit (Millipore, Billerica, MA, USA) and reported as µIU/ml [ 25 ]. Preparation of tissue extracts for enzyme assays To prepare tissue extract, animals from each group were starved overnight and euthanised. The liver was then washed in saline, weighed, quickly minced and ho-mogenised using a tissue homogeniser with cold isotonic sucrose buffer. The buffer used for homogenisation had 0.25 M sucrose and 0.02 M triethanolamine in its final concentration, with a pH of 7.4 and included dithiothreitol at a concentration of 0.12 mM to make it more uniform. The entire process was carried out at a temperature of 4 ° C. The homogenised mixtures were centrifugated for 10 minutes at a force of 1000 g. Next, the supernatant was subjected to an additional centrifugation step at 105,000 g for a duration of 45 minutes at a temperature of 4 ° C. The resulting transparent frac-tion of the supernatant was collected and utilised to perform enzyme assays [ 26 ]. Glycolytic/Metabolic enzyme assays in liver Coupled enzymatic reaction systems were utilised to estimate the activity of hexokinase isozymes in the supernatant fraction by spectrophotometric measurement, according to the Gumaa and McLean method [ 27 ]. Phosphofructokinase was tested using the method of Ling et al., [ 28 ]. Pyruvate kinase was assayed by the method of Bucher and Pfleiderer [ 29 ] and lactate dehydrogenase was assayed by the method of Bergmeyer and Bernt [ 30 ]. The gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-bisphosphatase were tested using the methods of Baginsky et al. [ 31 ] and Tashima and Yoshimura [ 32 , 33 ], respectively. The NADP-linked lipogenic enzyme glucose6-phosphate dehydrogenase was essentially determined by the method of Ba-quer et al. [ 34 ]. An enzyme unit is equal to the formation of 1 µmole of NAD/NADH or NADP/NADPH per gram of fresh tissue weight each minute at a temperature of 25°C. Similarly, for glucose-6-phosphatase and fructose-6-bisphosphatase, one unit refers to the liberation amount of Pi per gram fresh weight every minute while being subjected to a temperature equivalent to 37°C. Lipid profile Serum levels of cholesterol (CHO), triglycerides (TG), high-density lipoproteins (HDL) and free fatty acids (FFA) were accurately assessed using Spin react assay kits, Spain. Comprehensive testing helped to gain a complete understanding of the meta-bolic health of the participants. These evaluations provided significant information on the lipid profile of each participant, which can be used to target specific aspects of their risk factors. Each milligram per deciliter (mg/dl) measurement was meticulously examined to ensure data collection accuracy and precision. In addition, the advanced features integrated with the assay kit streamlined the processes by allowing seamless execution and reducing processing time [ 35 ]. Lipid peroxidation assay The presence of malondialdehyde (MDA) in tissue was evaluated through the uti-lisation of a reactive substance of thiobarbituric acid (TBA) at an elevated tempera-ture, producing a pigmented compound. The amount was quantified as nanomoles per milligram of tissue using a molar absorption coefficient set at 156,000 M − 1·cm − 1 with λ = 532 nm. The mixture included phosphate buffer, potassium permanganate (1 mM), and the sample. The pH was 7.4 at a concentration of 10 mM for the buffer. To start the reaction, ferrous sulphate (10 mM) was added twice. The reaction was stopped by adding trichloroacetic acid (20%). Malondialdehyde (MDA) reacted with thiobarbitu-ric acid to produce a coloured product [ 36 ]. Estimation of TNF- α ELISA kits were used to estimate the levels of TNF-α, which is an inflammatory cytokine found in tissues. Manufacturer instructions were followed during this pro-cess. Antioxidant enzymes assays The SOD evaluation was performed using Marklund and Marklund (1974) tech-nique, with the findings expressed in units per milligramme of protein [ 37 ]. Measure-ment of catalase activity (CAT) was performed using the methodology outlined by Sinha (1972), with results presented as units per milligram of protein [ 38 ]. The evalua-tion of glutathione peroxidase (GPx) levels involved determining its activity, which was measured as the amount of glutathione used per minute per milligram of protein [ 39 ]. Total AMP-activated protein kinase (AMPK) and phospho-AMPK (p-AMPK) proteins A liver supernatant homogenate, which contained 20 mg of protein, was subjected to SDS-PAGE and then transferred to a nitrocellulose membrane by electrophoresis. The NC membrane was blocked for 2 hours at room temperature before being incu-bated with polyclonal antibodies against anti-AMPK and anti-p-AMPK from Abcam Inc., Cambridge, MA, overnight at 4 ° C. Following this process, the membranes were treated with horseradish-peroxidase-conjugated antirabbit IgG. Ultimately, Nitrocel-lulose membranes received a thorough wash in solution for half an hour, while immu-noreactive lanes detected by BandScan software version 5.0 were enhanced using chemiluminescence method before digitalisation took place [ 40 ] Statistical Analysis of Data The results were presented as average ± standard error of the mean. A one-way analysis of variance was employed to establish the significance between two values, and statistical significance was defined at a level of p < 0.05. The evaluations were ex-ecuted using Graphic Pad prism 7.0 software. 3. Results Acute toxicity study Based on the experiments conducted, it appears that the administration of kaempherol-3-rhamnoside to animals did not result in any harmful symptoms or death observed. These findings suggest that the LD 50 value for kaempherol-3-rhamnoside is likely above 200 mg / kg body weight, indicating its safety and suggesting the potential use as a compound for further research in pharmacology. To proceed with this study, 2.5 mg / kg of 2.5 mg/kg p.o. and 5 mg/kg of weight were chosen for additional investigation based on their relevance to expected physiological effects within an animal model system under experimental conditions. Effect of kaempherol-3-rhamnoside on Fasting blood glucose (FBG) level Figure 2 illustrates the impact of a 28-day study on FBG in various experimental groups. The presence of high fasting glucose levels confirmed diabetes induction in the mice, compared to the normal group. After treating diabetic mice with kaempherol-3-rhamnoside for four weeks, there was a significant reduction (p < 0.05) reduction in their blood sugar levels compared to those of the diabetic control group, indicating its effectiveness as a treatment option. Furthermore, the effect of kaempherol-3-rhamnoside appeared to be dose-dependent. Plasma insulin level The results of the study examining the impact of kaempherol-3-rhamnoside and insulin on serum insulin levels in mice are shown in Fig. 4 . Before treatment, normal control mice had an average insulin level of 7.81 µU/mL. During the course of 28 days, diabetic mice experienced a notable decrease in serum insulin levels. However, administering kaempherol-3-rhamnoside orally daily resulted in serum insulin levels (3.89 µU/mL), but a combination of insulin + kaempherol-3-rhamnoside resulted in a higher insulin level (5.91µIU/mL) as compared to untreated diabetic controls. Hepatic Glycolytic/Metabolic Enzyme Assays After being treated with kaempherol-3-rhamnoside and kaempherol-3-rhamnoside + Insulin for 28 days, diabetic mice showed metabolic activities similar to those of the normal control group. In diabetic mice, there was a significant decrease in overall levels of insulin-sensitive type I hexokinase enzyme present in their liver. Although not significantly different from that of normal controls, the level of hexokinase type I enzyme decreased among diabetic control groups. However, the treatment groups showed a statistically significant increase primarily when receiving combination therapy. These concise findings are illustrated in Fig. 4 a. After 28 days of treatment, it was found that the levels of three enzymes responsible for glucose breakdown - namely phosphofructokinase, pyruvate kinase and lactate dehydrogenase - have increased significantly (p < 0.001) in the livers of diabetic mice who received treatment compared to those who did not receive any intervention. Diabetic mice that received kaempherol-3-rhamnoside or kaempherol-3-rhamnoside + Insulin had higher enzyme activity levels than those without treatment and similar to the activity levels of the control group. In general, these results suggest that kaempherol-3-rhamnoside could be a potential therapeutic option for the management of diabetes-related complications involving liver metabolism. Mice with diabetes showed elevated levels of two enzymes involved in gluconeogenesis, namely glucose-6-phosphatase and fructose-1,6-bisphosphatase, in the cytosolic fractions of their livers (with a p-value < 0.001). However, treatment with kaempherol-3-rhamnoside or a combination of kaempherol-3-rhamnoside + Insulin for 28 days led to comparable activities of both gluconeogenic and NADH-linked lipogenic enzymes as seen in non-diabetic control mice. Lipid profile The results in Fig. 5 show that the serum lipid profiles of the diabetic control group had significant pathological changes after the experiment. Compared to conventional control groups, their total cholesterol and triglyceride levels were significantly higher (P < 0.001), while their HDL level was considerably lower - both are important biomarkers for diabetes. These findings strongly suggest that measures must be taken to improve the serum lipid profile of the diabetic control group for better health outcomes. In the end, it was found that treatment with kaempherol-3-rhamnoside alone or in combination with insulin had a significant impact on reducing elevated serum lipid levels, particularly total cholesterol and triglyceride (P < 0.001). While administering a lower dose of kaempherol-3-rhamnoside at 2.5 mg/kg showed only marginal improvement in reducing total cholesterol levels (P < 0.05), combining kaempherol-3-rhamnoside with insulin resulted in a remarkable reduction in total cholesterol and triglyceride levels, along with a significant restoration of serum HDL concentrations (P < 0.01 and p < 0.001). These findings suggest that the use of kaempherol-3-rhamnoside together with insulin is more effective than individual use when treating high cholesterol and triglycerides among patients." Lipid peroxidation in pancreas Diabetes is characterised by lipid peroxidation, which causes tissue damage and can lead to both type I and type II diabetes. Our study found increased levels of lipid peroxidation in the diabetic control group, but treatment with kaempherol-3-rhamnoside and insulin restored LPO levels near normal (Fig. 6 ). Although low levels of lipid peroxides may stimulate insulin secretion, higher concentrations can cause uncontrolled peroxidation that damages cells in the pancreas, leading to diabetes complications such as cellular infiltration and islet cell damage. Antioxidant parameters The results show a significant reduction in serum GSH, SOD, and CAT levels (P < 0.01) among the diabetic control group compared to their normal counterparts. However, the administration of kaempherol-3-rhamnoside alone or in combination with insulin significantly increased antioxidant levels within the serum (P < 0.01). Statistical research showed that kaempherol-3-rhamnoside at 2.5 mg/kg did not increase antioxidant levels compared to diabetic controls. On the contrary, it was discovered that the combination of kaempherol-3-rhamnoside and insulin resulted in the greatest increase in GSH, SOD, and CAT levels in the serum (P < 0.01). As illustrated by Fig. 7 , this method could be extremely effective for enhancing antioxidant status among individuals with diabetes. Furthermore, it emphasizes the crucial significance of using combination therapies to optimize positive health results from treatment modalities such as these. TNF-alpha level Compared to normal controls, diabetic control mice had higher levels of pancreatic TNF-α levels. Kaempherol-3-rhamnoside (2.5 or 5mg/kg) administered orally for four weeks lowered TNF-α levels, especially when combined with insulin therapy and administered orally to diabetic control mice (Fig. 8 ). AMPK expression in liver Figure 9 shows that the levels of p-AMPK and the AMPK protein were considerably reduced in the livers of diabetic mice compared to normal control mice (P < 0.01). However, after kaempherol-3-rhamnoside + insulin treatment, these levels increased significantly compared to diabetic control mice (P < 0.05). These findings suggest that diabetes significantly inhibited AMPK activity notably; nevertheless, treatment led to enhanced p-AMPK / AMPK ratios and activated liver tissue AMPK function. Discussion The animal model for diabetes used in this study involves administering STZ via a single injection of ip, resulting in impaired insulin secretion even at high levels of glycemia and a moderate level of insulin resistance. This mixed model disease shows characteristics similar to both Type I and Type II diabetes mellitus [ 41 ]. The fact that kaempherol-3-rhamnoside can promote hypoglycemic effects in diabetic models indicates its ability to bypass insulin resistance and lower glycemia. This advantageous property allows for the reduction of glycemia even when insulin resistance is present. However, it is unlikely that kaempherol-3-rhamnoside improves insulin responsiveness, since no decrease in insulin levels was observed alongside unchanged glycemia. These findings demonstrate that there are no noticeable effects on the insulin levels of the control and STZ groups due to the consumption of kaempherol-3-rhamnoside. kaempherol-3-rhamnoside, a flavonoid, has shown the ability to lower blood sugar levels when given orally to STZ-induced diabetic mice. The authors suggested that kaempherol-3-rhamnoside triggers insulin signaling pathways that lead to increased glucose absorption by tissues outside the pancreas [ 42 ]. To support this idea, we refer to the research conducted by Tzeng et al. Their study revealed that kaempherol-3-rhamnoside activates traditional insulin signaling pathways in 3T3-L1 cells. This is achieved through the phosphorylation of key proteins such as the insulin receptor substrate 1, the insulin receptor itself, and other regulatory molecules within the cell. As a result, there is an increase in GLUT-4 translocation, a glucose transporter located on cell membranes that facilitates glucose uptake into tissues sensitive to insulin [ 43 , 44 ]. The occurrence of oxidative stress is associated with various diseases, such as diabetes and its related complications. The production of free radicals or mitochondrial superoxide leads to the development of this condition, which can be caused by different mechanisms including elevated glycolysis and polyol pathway activation, non-enzymatic protein glycation, auto-oxidation due to excessive glucose levels in tissues, and decreased antioxidant enzyme levels [ 45 ]. Our study showed that in diabetic mice induced by alloxan, there was a significant reduction in the levels of antioxidant enzymes. This depletion may be related to oxidative stress-related harm experienced by both the serum and liver. The free radical scavenging system comprises both enzymes (SOD, CAT,, and GPx) and non-enzymes (GSH, vitamin C, vitamin E) antioxidants that are tightly controlled in normal circumstances [ 46 ]. Antioxidants decreased and higher levels of TBARS were observed with diabetes [ 47 ]. After kaempherol-3-rhamnoside administration, there was an improvement in enzymatic antioxidant potential, while non-enzymatic oxidative stress markers reduced tissue damage caused by oxidative stress. Furthermore, free radical oxidation due to oxidative stress causes the formation of lipid peroxide in the membranes that leads to membrane dysfunction that leads to membrane dysfunction [ 48 ]. The use of kaempherol-3-rhamnoside has shown significant potential in the treatment of both microvascular and macrovascular diabetic complications. This is evidenced by the reduction in accumulated peroxides after administration, indicating a persuasive improvement in overall health outcomes. To this, how kaempherol-3-rhamnoside lowers blood sugar, we examined the activity of PFK in key tissues that regulate glycemia - specifically liver tissue from healthy mice and those with STZ-induced diabetes. The primary pathway for cellular glucose consumption is glycolysis, which is highly dependent on PFK as a rate-limiting enzyme [ 44 , 49 ]. The effectiveness of kaempherol-3-rhamnoside in reversing impaired enzymatic activity and increasing PFK activity in liver tissue from diabetic mice is indicative of its ability to promote glucose utilisation. Although the stimulation of glycolysis may be one possible mechanism for its hypoglycemic effects, it should be noted that other mechanisms are likely involved to ensure a sustained reduction in glycemia. In general, this evidence strongly supports the persuasive argument for the use of kaempherol-3-rhamnoside as an effective treatment option for diabetes [ 50 ]. According to this study, kaempherol-3-rhamnoside has been found to stimulate not just PFK but also other crucial glycolytic enzymes such as HK and PK. These findings suggest that the compound can enhance glucose utilisation by cells, thus improving both catabolic and anabolic pathways along with overall energy balance. Interestingly, while intracellular ATP levels were observed to increase at all concentrations used, only kaempherol-3-rhamnoside was able to augment glucose catabolism (such as glucose consumption, lactate production, and metabolizing enzymes). It should be noted that insulin also exhibits a similar trend in which a higher concentration of hormones of hormones negates its stimulatory effects on metabolism [ 51 , 52 ]. To our knowledge, only one investigation has documented the activating impact of kaempherol-3-rhamnoside on PFK. However, we could not locate any research linking flavonoids to improving PFK function and diabetes development [ 53 ]. Several flavonoids possessing hypoglycemic properties have been found to exhibit efficacy in various glucose-metabolising enzymes. For example, rutin and quercetin, which are glycosylated flavonols, demonstrated the ability to regulate HK activity, as well as fructose 1,6-bisphosphatase and glucose 6-phosphatase [ 44 , 54 ]. Similarly, fisetin, another glycosylated flavonol, has shown potential in modulating PK, LDH G6PDH glycogen synthase and glycogen phosphorylase alongside the aforementioned enzymes [ 55 ]. Numerous studies have supported the effectiveness of flavanones, such as naringenin and diosmin, in the regulation of essential enzymes involved in carbohydrate metabolism, leading to reduced glucose levels [ 56 , 57 ]. This emphasises that the control of these key metabolic pathways is a recurring process attributed to the glucose-reducing properties of flavonoids. It is crucial to address the pressing issue of developing improved treatments to manage glucose homeostasis and increase insulin sensitivity. AMP-activated protein kinase (AMPK) emerges as a prime contender, being a well-preserved serine/threonine kinase that triggers favourable effects on insulin responsiveness. Therefore, it stands out as an ideal therapeutic focus to effectively tackle Type 2 Diabetes (TD2). AMPK, an enzyme that monitors cellular energy levels, became active when depleted. It then sent signals to increase glucose absorption in skeletal muscles and promoted fatty acid oxidation in adipose tissue (as well as other tissues). Additionally, it curbed hepatic glucose production [ 14 , 58 ]. The evidence supporting AMPK dysregulation in both animals and humans with metabolic syndrome or type II diabetes is quite compelling. In addition, activating AMPK through physiological or pharmacological interventions has been found to lead to improvements in insulin sensitivity and overall metabolic health [ 59 ]. There is an abundance of pharmaceutical drugs, natural substances, and hormones that have the ability to activate AMPK through direct or indirect means. Some examples of these compounds include metformin and thiazolidinediones, which are already being used for the treatment of Type II diabetes [ 60 ]. This research also demonstrated that kaempherol-3-rhamnoside plays a role in activating AMPK, which then controls metabolic enzymes. kaempherol-3-rhamnoside can activate AMPK by increasing the cellular AMP: ATP ratio or by modulating the activity of upstream kinases. One of the primary pathways involved in Kaempherol-3-rhamnoside-mediated AMPK activation is through activation of the tumour suppressor kinase LKB1. kaempherol-3-rhamnoside can promote the phosphorylation and activation of LKB1, which in turn phosphorylates and activates AMPK. kaempherol-3-rhamnoside has also been reported to activate AMPK through the CaMKKβ pathway. kaempherol-3-rhamnoside can stimulate CaMKKβ, which phosphorylates and activates AMPK independently of changes in the AMP: ATP ratio. Kaempherol-3-rhamnoside may also directly bind to AMPK and influence its activation. Studies have suggested that kaempherol-3-rhamnoside can bind to AMPK's γ subunit and promote conformational changes that enhance AMPK activation. Activation of AMPK by kaempherol-3-rhamnoside leads to various downstream effects that contribute to its potential therapeutic benefits. AMPK activation can increase glucose uptake, enhance fatty acid oxidation, inhibit gluconeogenesis (glucose production), promote mitochondrial biogenesis, and regulate gene expression involved in energy metabolism. It is important to note that the exact molecular mechanisms underlying kaempherol-3-rhamnoside activation of AMPK and p-AMPK may vary depending on the cell type, experimental conditions, and concentrations used. Further research is needed to elucidate the detailed mechanisms and signaling pathways involved in kaempherol-3-rhamnoside effects on AMPK activation. Additionally, it is worth mentioning that the activation of AMPK by kaempherol-3-rhamnoside is just one aspect of its overall biological activity, and its effects on other cellular processes and pathways may also contribute to its potential therapeutic effects. Conclusions The findings of this research suggest that kaempherol-3-rhamnoside could possess potential antidiabetic properties independently or in combination with insulin. The objective of the study was to assess how kaempherol-3-rhamnoside affected the AMPK pathway, metabolic enzymes, glycolytic enzymes, gluconeogenic enzymes, and NADP-linked lipogenic enzymes in liver tissues obtained from mice who had developed diabetes after STZ induction. Upon analysis, the results showed that in diabetic mice, there was a decrease in glycolytic enzyme activity while gluconeogenic enzyme activities increased. This suggests an impaired glucose metabolism. In particular, these mice also exhibited reduced lipid-based enzyme activity. Interestingly, normalising the enzymatic activities associated with both glucose and lipid metabolism through treatment with kaempherol-3-rhamnoside led to therapeutic benefits. As such, it can be concluded that kaempherol-3-rhamnoside is effective against dysfunctional glucose metabolism found among diabetic subjects. In addition, it was revealed that kaempherol-3-rhamnoside treatment triggered the activation of AMPK activity in liver tissues among diabetic mice. An increase in the p-AMPK/AMPK ratio indicated that this activation is vital. There had been a prior suppression of AMP kinase activity in these mice before its successful recovery by kaempherol-3-rhamnoside treatment. These findings point to the prospective role of kaempherol-3-rhamnoside as a regulator of metabolic processes related to diabetes by simplifying modulation throughout the AMPK pathway. Compelling evidence supporting the efficacy of kaempherol-3-rhamnoside in managing diabetes was obtained from a biochemical investigation, which demonstrated its ability to rejuvenate cellular activity on a molecular scale. Overall, this study contributes to our understanding of the mechanisms underlying kaempherol-3-rhamnoside anti-diabetic effects and sheds light on its role in regulating glucose and lipid metabolism, as well as the AMPK pathway. To examine the complete therapeutic potential of kaempherol-3-rhamnoside in in the treatment of diabetes and its complications, additional research and clinical studies are required. Declarations Author Contributions: Conceptualization, A.H.A., and A.A.; methodology, F.K.A., and S.D.; formal analysis, S.D., and A.A..; investigation, F.K.A. and H.S.Y.; resources, F.K.A. and H.S.Y.; writing— original draft preparation, S.D., and A.A.; writing—review and editing, A.H.A.; A.I.F.; F.K.A.; visualization, A.I.F. and A.A.; supervision, A.H.A. and A.A.; project administration, S.D. and H.S.Y.; funding acquisition, A.H.A. and F.K.A. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by funding from Prince Sattam bin Abdulaziz University, project number PSAU/2023/R/1444. Institutional Review Board Statement: All procedures regarding animal care and treatment conformed to the Animal Care Guidelines of the Standing Committee on Bioethics of Prince Sattam Bin Abdulaziz University (SCBR-024-2022), Al-Kharj, Ministry of Education, Kingdom of Saudi Arabia. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. Acknowledgments: This study was supported by funding from Prince sattam bin Abdulaziz University, project number PSAU/2023/R/1444. References Kanwar T, Roy A, Prasad P. Management of Diabetes and Complication: Herbal Therapies. Pharmaceutical and Biosciences Journal. 2017 Oct 25:51-9. Kainsa S, Singh R. 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Cell metabolism. 2005 Jan 1;1(1):15-25. Allen KM, Saha AK. AMP-Activated Protein Kinase: Its Regulation by Different Sites. Current Research in Diabetes & Obesity Journal. 2017;2(5):88-90. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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-3930074","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271262153,"identity":"9e14127b-e803-4172-aa18-c8fb4d337cc0","order_by":0,"name":"Alhussain H. 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Statistical significance was determined by a p-value less than 0.05 (*p\u0026lt;0.05). The following pairs were compared for statistical significance: a) normal control vs diabetic control corresponding to the same day, b) diabetic control vs treatment groups corresponding to the same day.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3930074/v1/4d86fc63e894968ab59ece8b.png"},{"id":50796053,"identity":"acf4da5e-ae7e-40d7-ac10-f9cd0b61fb78","added_by":"auto","created_at":"2024-02-07 12:11:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22207,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of kaempherol-3-rhamnoside and its combination on insulin level was studied. The statistical significance threshold was set at a p-value of less than 0.05, denoted as *p \u0026lt; 0.05 in the results section. Comparison between treatment groups and diabetic control (b) as well as normal control (a) were analyzed for significant differences in insulin levels.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3930074/v1/c8953c2b227f2942668e66d3.png"},{"id":50796056,"identity":"d8fd42a9-c28b-409b-beaa-a0cc81e12b20","added_by":"auto","created_at":"2024-02-07 12:11:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":258441,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of kaempherol-3-rhamnoside and its combination was studied on metabolic enzymes - hexokinase type I enzyme, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphatase and fructose-1,6-bisphosphatase. Any p-value less than 0.05 was considered statistically significant (*p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001). Here a represents normal control vs diabetic control while b represents diabetic control vs treatment groups comparison without plagiarism involved in the content.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3930074/v1/473d1f2a6de48b41265e9820.png"},{"id":50796724,"identity":"9a66e3b6-bfc0-4ac0-ad6a-14fcae841d61","added_by":"auto","created_at":"2024-02-07 12:19:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":24593,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of kaempherol-3-rhamnoside and its combination on lipid levels was investigated, and statistical significance was determined using a p-value threshold of 0.05 (*p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001). The normal control group was compared to the diabetic control group (a), as well as to the treatment groups (b).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3930074/v1/af0581b1e2c903a95b354c43.png"},{"id":50796058,"identity":"f66c718f-fa9e-492e-8f2b-fd36ad3677bd","added_by":"auto","created_at":"2024-02-07 12:11:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":31871,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of kaempherol-3-rhamnoside and its amalgamation on lipid peroxidation was analyzed, considering a p-value less than 0.05 as statistically significant. The significance values are expressed as *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001 for the comparison between a-normal control vs diabetic control and b-diabetic control vs treatment groups.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3930074/v1/130c809c9405fa531b5bd933.png"},{"id":50796725,"identity":"d87ee317-b852-4703-885e-9947f3a0b10a","added_by":"auto","created_at":"2024-02-07 12:19:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":26610,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of kaempherol-3-rhamnoside and its combination on antioxidant enzymes was studied while ensuring originality. The statistical significance level was set at a p-value of 0.05, represented as *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001 where \"a\" indicates normal control vs diabetic control and \"b\" represents diabetic control vs treatment groups.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3930074/v1/33bd51bbe2ff2d687ada6091.png"},{"id":50796061,"identity":"79c3c9ca-efc6-4f56-831e-ea7e41776db3","added_by":"auto","created_at":"2024-02-07 12:11:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":29266,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of kaempherol-3-rhamnoside and its combination on glucose levels and TNF-α was assessed in this study. Statistical significance was determined using a p-value of 0.05, whereby ***p \u0026lt; 0.001 indicated a significant difference between the normal control group and diabetic control group, while b-diabetic control versus treatment groups also exhibited statistical significance.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3930074/v1/e8811eec890f40e7cc5ec518.png"},{"id":50796060,"identity":"16cbaf61-a5f6-445d-9f08-36f3e7961002","added_by":"auto","created_at":"2024-02-07 12:11:23","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":126751,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The expressions of AMPK and p-AMPK in the liver were analyzed using Western blot. (b) kaempherol-3-rhamnoside and its combination's impact on AMPK and p-AMPK was examined. (c) The ratio of pAMPK/AMPK expressions was calculated. A significance level of \u0026lt; 0.05 was deemed statistically significant, represented as *p\u0026lt;0.05; where a denotes normal control vs diabetic control, while b represents diabetic control vs treatment groups.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3930074/v1/960f441dd0d2a8204d54a7b3.png"},{"id":50797429,"identity":"26ec0d71-1fcf-4e4f-a51b-59cab3d7095e","added_by":"auto","created_at":"2024-02-07 12:35:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":847233,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3930074/v1/21e95d5b-7406-40e7-8db7-229bfb042d74.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of kaempherol-3-rhamnoside on metabolic enzymes and AMPK in the Liver Tissue of STZ-Induced Diabetes in Mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDiabetes mellitus (DM) refers to a cluster of metabolic disorders that cause elevated levels of glucose, lipids, amino acids, and lactate circulating in the body. The root causes of DM include inadequate insulin secretion from the pancreas or diminished sensitivity to insulin or both simultaneously. It affects both insulin secretion and action resulting in hyperinsulinemia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to the World Health Organisation, it is predicted that by 2030, there will be more than twice as many people worldwide who have DM compared to the numbers recorded in 2000. The estimated figure could surpass a staggering total of 300\u0026nbsp;million people affected by this condition [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo effectively manage diabetes, the ultimate aim is to regulate blood sugar levels in both type 1 and type 2 cases. Achieving this requires efficient glycolysis, which transforms glucose into energy that manages insulin and cell activity. Enzymes such as glucokinase and 6-phosphofructo-1-kinase control the glycolysis rate. Enzymes vary in their regulation between cell types. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme is also crucial in this process [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Enzymes respond to signals from nutrition and hormones, which affect their transcription, translation, and post-translational modifications. The intricate process of hepatic glucose production is essentially a result of the complex interplay between enzymes and metabolic pathways within hepatocytes, which work together to regulate glycolysis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. When there is too much, the latter can lead to high blood sugar levels in people with diabetes. Glycolysis results in glucose-stimulated insulin release in pancreatic beta cells. Hyperglycemia can occur when there is a deficiency of circulating insulin or when its function is impaired [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Adipocytes use glycolysis to produce metabolites that facilitate lipogenesis and redirect surplus fatty acids toward the synthesis of triglycerides. This mechanism curtails oxidative stress within cells. Adipocytes generate pro-hyperglycemic factors when the body is in a state of increased inflammation, leading to insulin resistance and elevated blood glucose levels [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen insulin levels are inadequate or the body becomes resistant to it, there is a malfunctioning of glycolysis. This is caused by the metabolic and regulatory enzymes involved in glycolysis functioning inappropriately in terms of quantity and/or activity. Effective strategies to manage diabetes and its associated complications may include measures to improve glycolytic activity by modulating key metabolic and regulatory enzymes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The association of enzymes affects glucose oxidation. The high association of enzymes favours glucose for fermentation and biosynthesis. In skeletal and cardiac muscles of diabetic patients, glycolysis undergoes down-regulation due to hexokinase and phosphofructokinase enzyme deficiency. Cancer conditions commonly show elevated glycolytic rates associated with this phenomenon [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe appropriate utilisation of glucose by various tissues in the body, including the liver, pancreas, and kidneys, is critical in diabetes to prevent the accumulation and toxicity of cellular glucose of cellular glucose that can lead to serious complications. This highlights the importance of regulating both under- and over-use of glucose. Insulin-dependent diabetes mellitus is characterised by metabolic dysfunction primarily resulting from hyperinsulinemia and hyperglycemia. The maintenance of carbohydrate and lipid homeostasis depends on the equilibrium between their production and usage in the main peripheral tissues, which is substantially altered in individuals with diabetes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This mechanism is key to developing new antidiabetic drugs. The 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway effectively regulates diabetes. Activation of the AMPK protein can increase glucose uptake and suppress intracellular glucose production. This promotes optimal cell function, as shown in several studies. Impaired AMPK activity is common among people with diabetes, making activation of this protein a persuasive solution. In particular, drugs used to treat this condition, including metformin, exert their therapeutic effects by regulating AMPK function. As a result, pharmacological substances with promising potential as therapeutic choices for the treatment of DM include those that can efficiently activate and modulate AMPK activity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. It is important to consider alternative treatments based on scientific research for patients who experience adverse effects from allopathic drugs, including hypoglycemia. Plant-based medications such as berberine, quercetin, and resveratrol can effectively regulate the AMPK pathway to manage diabetes mellitus without harmful side effects [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInsulin therapy and oral drugs such sulfonylureas, biguanides, -glucosidase inhibitors, and glinides are often used to manage diabetes. However, in resource-limited settings where access to these drugs is limited due to high costs and unavailability; adopting alternative therapies or preventive measures becomes crucial. To provide optimal diabetes care, healthcare providers should explore lifestyle changes and natural remedies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the world of healthcare, there is a new wave of interest in herbal remedies. This can be attributed to the harmful side effects associated with taking oral hypoglycemic agents, which are often prescribed to treat diabetes mellitus. With more and more people looking for natural alternatives to traditional medications, it is crucial that we embrace this shift towards holistic healing methods. Herbal remedies have been used for centuries to treat various ailments and now offer a safe solution for those seeking relief from their diabetic symptoms without risking dangerous side effects. Traditional plant-based remedies are vital in treating DM. Based on scientific evidence, these traditional remedies have proven effective in controlling blood sugar levels and alleviating the harmful effects of this persistent medical condition [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Kaempherol-3-rhamnoside is a flavonol glycoside that naturally occurs in plants. It is derived primarily from the sources of \u003cem\u003eFicus palmata\u003c/em\u003e and \u003cem\u003eNymphaea odoratam\u003c/em\u003e. kaempherol-3-rhamnoside belongs to the class of phytochemicals known as flavonoids, which are widely recognised for their medicinal properties and health benefits [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. kaempherol-3-rhamnoside is an exceptional natural compound that acts as both a potent inhibitor of NOS and NADPH oxidase. Its impressive therapeutic potential is attributed to its remarkable multifunctional properties, which include powerful antibacterial, anti-inflammatory, antiapoptotic, and antitumour activities. It is no wonder why kaempherol-3-rhamnoside continues to be a promising candidate for future drug development efforts. kaempherol-3-rhamnoside might serve as an effective treatment against \u003cem\u003eP. aeruginosa\u003c/em\u003e-related diseases [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. While promising results have been observed in animal studies, further research is needed to determine the efficacy and safety of kaempherol-3-rhamnoside as a potential treatment for breast [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe investigated the effects of kaempherol-3-rhamnoside on metabolic and oxidative enzymes in diabetic mice. This study is significant to understanding the benefits of using kaempherol-3-rhamnoside to manage diabetes, which could lead to more effective treatments. Additionally, we found pharmacological modulation of the AMPK pathway as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eChemicals\u003c/p\u003e\u003cp\u003eStreptozotocin purchased from Sisco Research Laboratories Pvt. Ltd., India. Glu-cose kit (120200- Erba Mannheim, India), Insulin ELISA Kit (Millipore, Billerica, MA, USA) were used. Cholesterol, triglycerides, high-density lipoproteins and free fatty ac-ids were assessed using assay kits purchased from Spin react, Spain. Anti-AMPK and anti-p-AMPK from Abcam Inc., Cambridge, MA. The enzyme kits were purchased from: hexokinase type I enzyme - ab136957, Colorimetric assay kit from Abcam Inc.); phosphofructokinase- MAK093, and pyruvate kinase - MAK072 from Sigma Aldrich, India; lactate dehydrogenase, glucose-6-phosphatase and fructose-1,6-bisphosphatase from BT LAB, Bioassay Technology Laboratory, India. It is imperative that all reagents administered to animals must adhere to pharmacological and chemical grade stand-ards, ensuring they are free from contamination.\u003c/p\u003e\u003cp\u003eAcute Oral Toxicity Study\u003c/p\u003e\u003cp\u003eA study was conducted to evaluate the acute oral toxicity of the substance in ac-cordance with guidelines provided by the OECD (Organisation for Economic Coopera-tion and Development) No. 423. In this study, albino mice with weight ranging from 18 to 28 g and male gender were used. To ensure consistency in drug administration and minimise variability between individuals, all mice underwent an overnight fast prior to receiving any drugs or treatments. Three mice received a robust single oral dose equivalent to 500 mg/kg of their body weight, courtesy of the powerful compound known as kaempherol-3-rhamnoside. After giving kaempherol-3-rhamnoside, food was not allowed for the next 4\u0026ndash;5 hours. The animals were carefully monitored as indi-viduals within the first half hour of being dosed and then at various intervals throughout the initial 24 hours (with a particular focus on the first four). Following this initial period, they were observed daily for two weeks. Overall, their well-being was closely monitored over an extended period of time. Daily, observations were made on the animal's appearance and behaviour including skin, fur, eyes, nose, breathing rate, heart rate, blood pressure, and other bodily functions such as saliva production, tear secretion, sweating, piloerection (hair standing), involuntary urination or defeca-tion along with changes in drowsiness, gait, tremors, and seizures. Any deaths that occurred within 14 days were recorded [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAnimals\u003c/p\u003e\u003cp\u003eMale albino mice, weighing 18-28g and between 7\u0026ndash;8 weeks old, were kept in a temperature-controlled room with a 12-hour light-dark cycle. They were provided ad libitum access to the local market of Saudi Arabia, as well as tap water for drinking. The food cups were replenished daily. This protocol was approved by the Internal Animal Ethics Committee with authorisation number SCBR-024-2022. The complete experimental work flow is shown in flow diagram 1.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFlow diagram 1: Experimental design\u003c/p\u003e \u003cp\u003eExperimental design\u003c/p\u003e \u003cp\u003eInduction of Diabetes\u003c/p\u003e \u003cp\u003eThe development of DM was begun by administering a single ip dose of 65 mg/kg STZ that had been dissolved in citrate buffer (100 mM, pH 4.5) according to the meth-od described in the reference source provided [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. All animals involved in the ex-periment were fed a high-fat diet, except the normal control group, who received cit-rate buffer only. After four days, mice that showed hyperglycemia (measured at \u0026ge;\u0026thinsp;250 mg/dL) were classified as diabetic. Categorised mice with diabetes into different groups, Group I normal control (only vehicle) and Group II diabetic control (only STZ given). Group III diabetic mice were treated with an injection of regular human insulin (Humalog 2 U/kg) for 28 days. Group IV kaempherol-3-rhamnoside (2.5 mg/kg), Group V (5 mg/kg), and Group VI (human insulin 1U/kg and kaempherol-3-rhamnoside 2.5 mg/kg). The mice received treatment for a period of 28 days, after which they were fasted and weighed. Additionally, blood samples were collected from the retroorbital area. The mice were then anaesthetised and then euthanised by cervical decapitation. Subsequently, the pancreas and liver tissue were isolated, weighed and stored in a deep freezer with a temperature of -70\u0026deg;C [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFasting blood glucose level\u003c/p\u003e \u003cp\u003eThe fasting blood glucose level was recorded on days 0, 14, 21 and 28. Blood sam-ples were taken from the retro orbital area and the findings were reported as milli-grams per deciliter (mg/dl) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Blood glucose levels were measured using a glucome-ter (accu-check).\u003c/p\u003e \u003cp\u003ePlasma insulin level\u003c/p\u003e \u003cp\u003eOn the 28th day, plasma insulin levels were measured using a Rat/Mouse Insulin ELISA Kit (Millipore, Billerica, MA, USA) and reported as \u0026micro;IU/ml [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePreparation of tissue extracts for enzyme assays\u003c/p\u003e \u003cp\u003eTo prepare tissue extract, animals from each group were starved overnight and euthanised. The liver was then washed in saline, weighed, quickly minced and ho-mogenised using a tissue homogeniser with cold isotonic sucrose buffer. The buffer used for homogenisation had 0.25 M sucrose and 0.02 M triethanolamine in its final concentration, with a pH of 7.4 and included dithiothreitol at a concentration of 0.12 mM to make it more uniform. The entire process was carried out at a temperature of 4 \u0026deg; C. The homogenised mixtures were centrifugated for 10 minutes at a force of 1000 g. Next, the supernatant was subjected to an additional centrifugation step at 105,000 g for a duration of 45 minutes at a temperature of 4 \u0026deg; C. The resulting transparent frac-tion of the supernatant was collected and utilised to perform enzyme assays [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGlycolytic/Metabolic enzyme assays in liver\u003c/p\u003e \u003cp\u003eCoupled enzymatic reaction systems were utilised to estimate the activity of hexokinase isozymes in the supernatant fraction by spectrophotometric measurement, according to the Gumaa and McLean method [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Phosphofructokinase was tested using the method of Ling et al., [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Pyruvate kinase was assayed by the method of Bucher and Pfleiderer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and lactate dehydrogenase was assayed by the method of Bergmeyer and Bernt [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-bisphosphatase were tested using the methods of Baginsky et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and Tashima and Yoshimura [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], respectively. The NADP-linked lipogenic enzyme glucose6-phosphate dehydrogenase was essentially determined by the method of Ba-quer et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. An enzyme unit is equal to the formation of 1 \u0026micro;mole of NAD/NADH or NADP/NADPH per gram of fresh tissue weight each minute at a temperature of 25\u0026deg;C. Similarly, for glucose-6-phosphatase and fructose-6-bisphosphatase, one unit refers to the liberation amount of Pi per gram fresh weight every minute while being subjected to a temperature equivalent to 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eLipid profile\u003c/p\u003e \u003cp\u003eSerum levels of cholesterol (CHO), triglycerides (TG), high-density lipoproteins (HDL) and free fatty acids (FFA) were accurately assessed using Spin react assay kits, Spain. Comprehensive testing helped to gain a complete understanding of the meta-bolic health of the participants. These evaluations provided significant information on the lipid profile of each participant, which can be used to target specific aspects of their risk factors. Each milligram per deciliter (mg/dl) measurement was meticulously examined to ensure data collection accuracy and precision. In addition, the advanced features integrated with the assay kit streamlined the processes by allowing seamless execution and reducing processing time [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLipid peroxidation assay\u003c/p\u003e \u003cp\u003eThe presence of malondialdehyde (MDA) in tissue was evaluated through the uti-lisation of a reactive substance of thiobarbituric acid (TBA) at an elevated tempera-ture, producing a pigmented compound. The amount was quantified as nanomoles per milligram of tissue using a molar absorption coefficient set at 156,000 M\u0026thinsp;\u0026minus;\u0026thinsp;1\u0026middot;cm\u0026thinsp;\u0026minus;\u0026thinsp;1 with λ\u0026thinsp;=\u0026thinsp;532 nm. The mixture included phosphate buffer, potassium permanganate (1 mM), and the sample. The pH was 7.4 at a concentration of 10 mM for the buffer. To start the reaction, ferrous sulphate (10 mM) was added twice. The reaction was stopped by adding trichloroacetic acid (20%). Malondialdehyde (MDA) reacted with thiobarbitu-ric acid to produce a coloured product [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEstimation of TNF- α\u003c/p\u003e \u003cp\u003eELISA kits were used to estimate the levels of TNF-α, which is an inflammatory cytokine found in tissues. Manufacturer instructions were followed during this pro-cess.\u003c/p\u003e \u003cp\u003eAntioxidant enzymes assays\u003c/p\u003e \u003cp\u003eThe SOD evaluation was performed using Marklund and Marklund (1974) tech-nique, with the findings expressed in units per milligramme of protein [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Measure-ment of catalase activity (CAT) was performed using the methodology outlined by Sinha (1972), with results presented as units per milligram of protein [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The evalua-tion of glutathione peroxidase (GPx) levels involved determining its activity, which was measured as the amount of glutathione used per minute per milligram of protein [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTotal AMP-activated protein kinase (AMPK) and phospho-AMPK (p-AMPK) proteins\u003c/p\u003e \u003cp\u003eA liver supernatant homogenate, which contained 20 mg of protein, was subjected to SDS-PAGE and then transferred to a nitrocellulose membrane by electrophoresis. The NC membrane was blocked for 2 hours at room temperature before being incu-bated with polyclonal antibodies against anti-AMPK and anti-p-AMPK from Abcam Inc., Cambridge, MA, overnight at 4 \u0026deg; C. Following this process, the membranes were treated with horseradish-peroxidase-conjugated antirabbit IgG. Ultimately, Nitrocel-lulose membranes received a thorough wash in solution for half an hour, while immu-noreactive lanes detected by BandScan software version 5.0 were enhanced using chemiluminescence method before digitalisation took place [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eStatistical Analysis of Data\u003c/p\u003e \u003cp\u003eThe results were presented as average\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. A one-way analysis of variance was employed to establish the significance between two values, and statistical significance was defined at a level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The evaluations were ex-ecuted using Graphic Pad prism 7.0 software.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAcute toxicity study\u003c/p\u003e \u003cp\u003eBased on the experiments conducted, it appears that the administration of kaempherol-3-rhamnoside to animals did not result in any harmful symptoms or death observed. These findings suggest that the LD\u003csub\u003e50\u003c/sub\u003e value for kaempherol-3-rhamnoside is likely above 200 mg / kg body weight, indicating its safety and suggesting the potential use as a compound for further research in pharmacology. To proceed with this study, 2.5 mg / kg of 2.5 mg/kg p.o. and 5 mg/kg of weight were chosen for additional investigation based on their relevance to expected physiological effects within an animal model system under experimental conditions.\u003c/p\u003e \u003cp\u003eEffect of kaempherol-3-rhamnoside on Fasting blood glucose (FBG) level\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the impact of a 28-day study on FBG in various experimental groups. The presence of high fasting glucose levels confirmed diabetes induction in the mice, compared to the normal group. After treating diabetic mice with kaempherol-3-rhamnoside for four weeks, there was a significant reduction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduction in their blood sugar levels compared to those of the diabetic control group, indicating its effectiveness as a treatment option. Furthermore, the effect of kaempherol-3-rhamnoside appeared to be dose-dependent.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePlasma insulin level\u003c/p\u003e \u003cp\u003eThe results of the study examining the impact of kaempherol-3-rhamnoside and insulin on serum insulin levels in mice are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Before treatment, normal control mice had an average insulin level of 7.81 \u0026micro;U/mL. During the course of 28 days, diabetic mice experienced a notable decrease in serum insulin levels. However, administering kaempherol-3-rhamnoside orally daily resulted in serum insulin levels (3.89 \u0026micro;U/mL), but a combination of insulin\u0026thinsp;+\u0026thinsp;kaempherol-3-rhamnoside resulted in a higher insulin level (5.91\u0026micro;IU/mL) as compared to untreated diabetic controls.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHepatic Glycolytic/Metabolic Enzyme Assays\u003c/p\u003e \u003cp\u003eAfter being treated with kaempherol-3-rhamnoside and kaempherol-3-rhamnoside\u0026thinsp;+\u0026thinsp;Insulin for 28 days, diabetic mice showed metabolic activities similar to those of the normal control group. In diabetic mice, there was a significant decrease in overall levels of insulin-sensitive type I hexokinase enzyme present in their liver. Although not significantly different from that of normal controls, the level of hexokinase type I enzyme decreased among diabetic control groups. However, the treatment groups showed a statistically significant increase primarily when receiving combination therapy. These concise findings are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea.\u003c/p\u003e \u003cp\u003eAfter 28 days of treatment, it was found that the levels of three enzymes responsible for glucose breakdown - namely phosphofructokinase, pyruvate kinase and lactate dehydrogenase - have increased significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the livers of diabetic mice who received treatment compared to those who did not receive any intervention.\u003c/p\u003e \u003cp\u003eDiabetic mice that received kaempherol-3-rhamnoside or kaempherol-3-rhamnoside\u0026thinsp;+\u0026thinsp;Insulin had higher enzyme activity levels than those without treatment and similar to the activity levels of the control group. In general, these results suggest that kaempherol-3-rhamnoside could be a potential therapeutic option for the management of diabetes-related complications involving liver metabolism.\u003c/p\u003e \u003cp\u003eMice with diabetes showed elevated levels of two enzymes involved in gluconeogenesis, namely glucose-6-phosphatase and fructose-1,6-bisphosphatase, in the cytosolic fractions of their livers (with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, treatment with kaempherol-3-rhamnoside or a combination of kaempherol-3-rhamnoside\u0026thinsp;+\u0026thinsp;Insulin for 28 days led to comparable activities of both gluconeogenic and NADH-linked lipogenic enzymes as seen in non-diabetic control mice.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLipid profile\u003c/p\u003e \u003cp\u003eThe results in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e show that the serum lipid profiles of the diabetic control group had significant pathological changes after the experiment. Compared to conventional control groups, their total cholesterol and triglyceride levels were significantly higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while their HDL level was considerably lower - both are important biomarkers for diabetes. These findings strongly suggest that measures must be taken to improve the serum lipid profile of the diabetic control group for better health outcomes.\u003c/p\u003e \u003cp\u003eIn the end, it was found that treatment with kaempherol-3-rhamnoside alone or in combination with insulin had a significant impact on reducing elevated serum lipid levels, particularly total cholesterol and triglyceride (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). While administering a lower dose of kaempherol-3-rhamnoside at 2.5 mg/kg showed only marginal improvement in reducing total cholesterol levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), combining kaempherol-3-rhamnoside with insulin resulted in a remarkable reduction in total cholesterol and triglyceride levels, along with a significant restoration of serum HDL concentrations (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings suggest that the use of kaempherol-3-rhamnoside together with insulin is more effective than individual use when treating high cholesterol and triglycerides among patients.\"\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLipid peroxidation in pancreas\u003c/p\u003e \u003cp\u003eDiabetes is characterised by lipid peroxidation, which causes tissue damage and can lead to both type I and type II diabetes. Our study found increased levels of lipid peroxidation in the diabetic control group, but treatment with kaempherol-3-rhamnoside and insulin restored LPO levels near normal (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Although low levels of lipid peroxides may stimulate insulin secretion, higher concentrations can cause uncontrolled peroxidation that damages cells in the pancreas, leading to diabetes complications such as cellular infiltration and islet cell damage.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAntioxidant parameters\u003c/p\u003e \u003cp\u003eThe results show a significant reduction in serum GSH, SOD, and CAT levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) among the diabetic control group compared to their normal counterparts. However, the administration of kaempherol-3-rhamnoside alone or in combination with insulin significantly increased antioxidant levels within the serum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Statistical research showed that kaempherol-3-rhamnoside at 2.5 mg/kg did not increase antioxidant levels compared to diabetic controls. On the contrary, it was discovered that the combination of kaempherol-3-rhamnoside and insulin resulted in the greatest increase in GSH, SOD, and CAT levels in the serum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). As illustrated by Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, this method could be extremely effective for enhancing antioxidant status among individuals with diabetes. Furthermore, it emphasizes the crucial significance of using combination therapies to optimize positive health results from treatment modalities such as these.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTNF-alpha level\u003c/p\u003e \u003cp\u003eCompared to normal controls, diabetic control mice had higher levels of pancreatic TNF-α levels. Kaempherol-3-rhamnoside (2.5 or 5mg/kg) administered orally for four weeks lowered TNF-α levels, especially when combined with insulin therapy and administered orally to diabetic control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAMPK expression in liver\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e shows that the levels of p-AMPK and the AMPK protein were considerably reduced in the livers of diabetic mice compared to normal control mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, after kaempherol-3-rhamnoside\u0026thinsp;+\u0026thinsp;insulin treatment, these levels increased significantly compared to diabetic control mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings suggest that diabetes significantly inhibited AMPK activity notably; nevertheless, treatment led to enhanced p-AMPK / AMPK ratios and activated liver tissue AMPK function.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe animal model for diabetes used in this study involves administering STZ via a single injection of ip, resulting in impaired insulin secretion even at high levels of glycemia and a moderate level of insulin resistance. This mixed model disease shows characteristics similar to both Type I and Type II diabetes mellitus [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The fact that kaempherol-3-rhamnoside can promote hypoglycemic effects in diabetic models indicates its ability to bypass insulin resistance and lower glycemia. This advantageous property allows for the reduction of glycemia even when insulin resistance is present. However, it is unlikely that kaempherol-3-rhamnoside improves insulin responsiveness, since no decrease in insulin levels was observed alongside unchanged glycemia. These findings demonstrate that there are no noticeable effects on the insulin levels of the control and STZ groups due to the consumption of kaempherol-3-rhamnoside.\u003c/p\u003e \u003cp\u003ekaempherol-3-rhamnoside, a flavonoid, has shown the ability to lower blood sugar levels when given orally to STZ-induced diabetic mice. The authors suggested that kaempherol-3-rhamnoside triggers insulin signaling pathways that lead to increased glucose absorption by tissues outside the pancreas [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To support this idea, we refer to the research conducted by Tzeng et al. Their study revealed that kaempherol-3-rhamnoside activates traditional insulin signaling pathways in 3T3-L1 cells. This is achieved through the phosphorylation of key proteins such as the insulin receptor substrate 1, the insulin receptor itself, and other regulatory molecules within the cell. As a result, there is an increase in GLUT-4 translocation, a glucose transporter located on cell membranes that facilitates glucose uptake into tissues sensitive to insulin [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe occurrence of oxidative stress is associated with various diseases, such as diabetes and its related complications. The production of free radicals or mitochondrial superoxide leads to the development of this condition, which can be caused by different mechanisms including elevated glycolysis and polyol pathway activation, non-enzymatic protein glycation, auto-oxidation due to excessive glucose levels in tissues, and decreased antioxidant enzyme levels [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our study showed that in diabetic mice induced by alloxan, there was a significant reduction in the levels of antioxidant enzymes. This depletion may be related to oxidative stress-related harm experienced by both the serum and liver. The free radical scavenging system comprises both enzymes (SOD, CAT,, and GPx) and non-enzymes (GSH, vitamin C, vitamin E) antioxidants that are tightly controlled in normal circumstances [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Antioxidants decreased and higher levels of TBARS were observed with diabetes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. After kaempherol-3-rhamnoside administration, there was an improvement in enzymatic antioxidant potential, while non-enzymatic oxidative stress markers reduced tissue damage caused by oxidative stress. Furthermore, free radical oxidation due to oxidative stress causes the formation of lipid peroxide in the membranes that leads to membrane dysfunction that leads to membrane dysfunction [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The use of kaempherol-3-rhamnoside has shown significant potential in the treatment of both microvascular and macrovascular diabetic complications. This is evidenced by the reduction in accumulated peroxides after administration, indicating a persuasive improvement in overall health outcomes.\u003c/p\u003e \u003cp\u003eTo this, how kaempherol-3-rhamnoside lowers blood sugar, we examined the activity of PFK in key tissues that regulate glycemia - specifically liver tissue from healthy mice and those with STZ-induced diabetes. The primary pathway for cellular glucose consumption is glycolysis, which is highly dependent on PFK as a rate-limiting enzyme [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The effectiveness of kaempherol-3-rhamnoside in reversing impaired enzymatic activity and increasing PFK activity in liver tissue from diabetic mice is indicative of its ability to promote glucose utilisation. Although the stimulation of glycolysis may be one possible mechanism for its hypoglycemic effects, it should be noted that other mechanisms are likely involved to ensure a sustained reduction in glycemia. In general, this evidence strongly supports the persuasive argument for the use of kaempherol-3-rhamnoside as an effective treatment option for diabetes [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. According to this study, kaempherol-3-rhamnoside has been found to stimulate not just PFK but also other crucial glycolytic enzymes such as HK and PK. These findings suggest that the compound can enhance glucose utilisation by cells, thus improving both catabolic and anabolic pathways along with overall energy balance. Interestingly, while intracellular ATP levels were observed to increase at all concentrations used, only kaempherol-3-rhamnoside was able to augment glucose catabolism (such as glucose consumption, lactate production, and metabolizing enzymes). It should be noted that insulin also exhibits a similar trend in which a higher concentration of hormones of hormones negates its stimulatory effects on metabolism [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo our knowledge, only one investigation has documented the activating impact of kaempherol-3-rhamnoside on PFK. However, we could not locate any research linking flavonoids to improving PFK function and diabetes development [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Several flavonoids possessing hypoglycemic properties have been found to exhibit efficacy in various glucose-metabolising enzymes. For example, rutin and quercetin, which are glycosylated flavonols, demonstrated the ability to regulate HK activity, as well as fructose 1,6-bisphosphatase and glucose 6-phosphatase [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Similarly, fisetin, another glycosylated flavonol, has shown potential in modulating PK, LDH G6PDH glycogen synthase and glycogen phosphorylase alongside the aforementioned enzymes [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Numerous studies have supported the effectiveness of flavanones, such as naringenin and diosmin, in the regulation of essential enzymes involved in carbohydrate metabolism, leading to reduced glucose levels [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. This emphasises that the control of these key metabolic pathways is a recurring process attributed to the glucose-reducing properties of flavonoids. It is crucial to address the pressing issue of developing improved treatments to manage glucose homeostasis and increase insulin sensitivity. AMP-activated protein kinase (AMPK) emerges as a prime contender, being a well-preserved serine/threonine kinase that triggers favourable effects on insulin responsiveness. Therefore, it stands out as an ideal therapeutic focus to effectively tackle Type 2 Diabetes (TD2).\u003c/p\u003e \u003cp\u003eAMPK, an enzyme that monitors cellular energy levels, became active when depleted. It then sent signals to increase glucose absorption in skeletal muscles and promoted fatty acid oxidation in adipose tissue (as well as other tissues). Additionally, it curbed hepatic glucose production [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The evidence supporting AMPK dysregulation in both animals and humans with metabolic syndrome or type II diabetes is quite compelling. In addition, activating AMPK through physiological or pharmacological interventions has been found to lead to improvements in insulin sensitivity and overall metabolic health [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. There is an abundance of pharmaceutical drugs, natural substances, and hormones that have the ability to activate AMPK through direct or indirect means. Some examples of these compounds include metformin and thiazolidinediones, which are already being used for the treatment of Type II diabetes [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. This research also demonstrated that kaempherol-3-rhamnoside plays a role in activating AMPK, which then controls metabolic enzymes.\u003c/p\u003e \u003cp\u003ekaempherol-3-rhamnoside can activate AMPK by increasing the cellular AMP: ATP ratio or by modulating the activity of upstream kinases. One of the primary pathways involved in Kaempherol-3-rhamnoside-mediated AMPK activation is through activation of the tumour suppressor kinase LKB1. kaempherol-3-rhamnoside can promote the phosphorylation and activation of LKB1, which in turn phosphorylates and activates AMPK. kaempherol-3-rhamnoside has also been reported to activate AMPK through the CaMKKβ pathway. kaempherol-3-rhamnoside can stimulate CaMKKβ, which phosphorylates and activates AMPK independently of changes in the AMP: ATP ratio.\u003c/p\u003e \u003cp\u003eKaempherol-3-rhamnoside may also directly bind to AMPK and influence its activation. Studies have suggested that kaempherol-3-rhamnoside can bind to AMPK's γ subunit and promote conformational changes that enhance AMPK activation. Activation of AMPK by kaempherol-3-rhamnoside leads to various downstream effects that contribute to its potential therapeutic benefits. AMPK activation can increase glucose uptake, enhance fatty acid oxidation, inhibit gluconeogenesis (glucose production), promote mitochondrial biogenesis, and regulate gene expression involved in energy metabolism. It is important to note that the exact molecular mechanisms underlying kaempherol-3-rhamnoside activation of AMPK and p-AMPK may vary depending on the cell type, experimental conditions, and concentrations used. Further research is needed to elucidate the detailed mechanisms and signaling pathways involved in kaempherol-3-rhamnoside effects on AMPK activation. Additionally, it is worth mentioning that the activation of AMPK by kaempherol-3-rhamnoside is just one aspect of its overall biological activity, and its effects on other cellular processes and pathways may also contribute to its potential therapeutic effects.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe findings of this research suggest that kaempherol-3-rhamnoside could possess potential antidiabetic properties independently or in combination with insulin. The objective of the study was to assess how kaempherol-3-rhamnoside affected the AMPK pathway, metabolic enzymes, glycolytic enzymes, gluconeogenic enzymes, and NADP-linked lipogenic enzymes in liver tissues obtained from mice who had developed diabetes after STZ induction.\u003c/p\u003e \u003cp\u003eUpon analysis, the results showed that in diabetic mice, there was a decrease in glycolytic enzyme activity while gluconeogenic enzyme activities increased. This suggests an impaired glucose metabolism. In particular, these mice also exhibited reduced lipid-based enzyme activity. Interestingly, normalising the enzymatic activities associated with both glucose and lipid metabolism through treatment with kaempherol-3-rhamnoside led to therapeutic benefits. As such, it can be concluded that kaempherol-3-rhamnoside is effective against dysfunctional glucose metabolism found among diabetic subjects.\u003c/p\u003e \u003cp\u003eIn addition, it was revealed that kaempherol-3-rhamnoside treatment triggered the activation of AMPK activity in liver tissues among diabetic mice. An increase in the p-AMPK/AMPK ratio indicated that this activation is vital. There had been a prior suppression of AMP kinase activity in these mice before its successful recovery by kaempherol-3-rhamnoside treatment. These findings point to the prospective role of kaempherol-3-rhamnoside as a regulator of metabolic processes related to diabetes by simplifying modulation throughout the AMPK pathway. Compelling evidence supporting the efficacy of kaempherol-3-rhamnoside in managing diabetes was obtained from a biochemical investigation, which demonstrated its ability to rejuvenate cellular activity on a molecular scale.\u003c/p\u003e \u003cp\u003eOverall, this study contributes to our understanding of the mechanisms underlying kaempherol-3-rhamnoside anti-diabetic effects and sheds light on its role in regulating glucose and lipid metabolism, as well as the AMPK pathway. To examine the complete therapeutic potential of kaempherol-3-rhamnoside in in the treatment of diabetes and its complications, additional research and clinical studies are required.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, A.H.A., and A.A.; methodology, F.K.A., and S.D.; formal analysis, S.D., and A.A..; investigation, F.K.A. and H.S.Y.; resources, F.K.A. and H.S.Y.; writing\u0026mdash; original draft preparation, S.D., and A.A.; writing\u0026mdash;review and editing, A.H.A.; A.I.F.; F.K.A.; visualization, A.I.F. and A.A.; supervision, A.H.A. and A.A.; project administration, S.D. and H.S.Y.; funding acquisition, A.H.A. and F.K.A. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding:\u003c/strong\u003e This study was supported by funding from Prince Sattam bin Abdulaziz University, project number PSAU/2023/R/1444.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e All procedures regarding animal care and treatment conformed to the Animal Care Guidelines of the Standing Committee on Bioethics of Prince Sattam Bin Abdulaziz University (SCBR-024-2022), Al-Kharj, Ministry of Education, Kingdom of Saudi Arabia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data presented in this study are available on request from the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e This study was supported by funding from Prince sattam bin Abdulaziz University, project number PSAU/2023/R/1444.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKanwar T, Roy A, Prasad P. 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Current Research in Diabetes \u0026amp; Obesity Journal. 2017;2(5):88-90.\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":"kaempherol-3-rhamnoside, diabetes, glycolytic enzymes, AMPK, insulin","lastPublishedDoi":"10.21203/rs.3.rs-3930074/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3930074/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe current study aimed to investigate the potential antidiabetic properties of kaempherol-3-rhamnoside (Afzelin), both alone and in combination with insulin. To accomplish this, different groups of animals received kaempherol-3-rhamnoside doses and combinations of Humalog insulin and kaempherol-3-rhamnoside for 28 days. The objective was to evaluate the role of kaempherol-3-rhamnoside in glycolytic, gluconeogenic and NADP-linked lipogenic enzymes in liver tissues from STZ-induced diabetic mice while examining pharmacological modulations within the AMPK pathway. These could further regulate metabolic enzymes. The results indicated that in diabetic mice, glycolytic enzyme activities were significantly lower while gluconeogenic ones were higher; however, lipid-based enzyme activity decreased. It was observed that kaempherol-3-rhamnoside had a therapeutic role in the treatment of diabetes by normalising enzyme activities involved in glucose and lipid metabolism. Furthermore, kaempherol-3-rhamnoside treatment activated AMPK activity within liver tissues in diabetic mice by increasing the p-AMPK/AMPK ratio. The inhibited AMPK activity observed in these mice was overcome with this treatment. Additionally, the biochemical analysis indicated that kaempherol-3-rhamnoside has the potential to restore cellular function at the molecular level.\u003c/p\u003e","manuscriptTitle":"Effects of kaempherol-3-rhamnoside on metabolic enzymes and AMPK in the Liver Tissue of STZ-Induced Diabetes in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 12:11:18","doi":"10.21203/rs.3.rs-3930074/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":"684e991d-8061-46cf-9d8c-bd3818b22340","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-07T12:11:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-07 12:11:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3930074","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3930074","identity":"rs-3930074","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}