The International Diabetes Federation (IDF) reports that diabetes mellitus continues to pose as a major global health issue because it affects more than 537 million adults across the world in 2021 while projections indicate that this number will rise to 783 million by 2045 (International Diabetes Federation, 2021) [1]. Chronic hyperglycemia leads to severe complications that cause most deaths among diabetic patients. To enhance therapeutic outcomes, it is essential to comprehend the metabolic processes that lead to diabetes progression.

The liver functions as a vital organ which executes the detoxification of both xenobiotics and endogenous substances [2]. The liver's detoxification work is highly individualized because it depends on the endoplasmic reticulum-based microsomal monooxygenase system activity in hepatocytes [3]. Cytochrome P450 (CYP450), as the essential component of this system to metabolize exogenous compounds and drugs together with environmental toxins and dietary components while also metabolizing endogenous substances including steroid hormones and fatty acids [4][5].

The CYP450-dependent monooxygenase system displays functional variability through genetic and phenotypic elements. Human CYP450 activity produces phenotypic differences which result in three distinct metabolic categories: "slow", "rapid" and "ultrarapid" metabolizers [6]. A person with no functioning alleles demonstrates "poor" metabolism while extensive metabolism occurs in individuals who have one to two functioning alleles and rapid metabolism is observed in people who have more than two alleles. Modern pharmacogenetic tests enable researchers to determine human genotypes outside the body without invasive procedures.

Human beings experience altered phenotypic expressions because of external influences from different chemicals, medications and untypical foods. H.P. Sheldon and colleagues performed a detailed examination of how treatment affected 900 depression patients through phenotypic modification (7). When the research began patients received a genotype classification of "poor" metabolism in 3.9% of participants while "rapid" and "ultrarapid" metabolism classifications accounted for 96.1% of patients. Following the treatment phase the patient population shifted to include 27% poor metabolizers and 67% rapid and ultrarapid metabolizers according to phenotypic classification. The research shows that 23% of patients displayed poor metabolism phenotypes even though their genotype initially indicated rapid or ultrarapid metabolism.

The distinction between genotype and phenotype demonstrates much lower variation among animals compared to human beings. Metabolic activity creates substantial variations in physiological and pathological processes between members of the same species. The metabolic variations among patients become crucial in diabetes mellitus since metabolic imbalances tend to worsen disease progression. Rats along with other animal models enable researchers to examine these biological mechanisms inside laboratory settings. The functional activity of liver detoxification systems produces variations in biochemical markers and physiological parameters which reveal metabolic status information about the organism.

The experimental model of alloxan-induced diabetes serves as an established method to study the pathophysiology of diabetes mellitus. The pancreatic β-cells become specifically damaged by alloxan which causes elevated blood glucose levels and metabolic problems. Research has failed to fully explain the relationship between liver detoxification functional activity and the development of alloxan-induced diabetes [8].

The functional condition of liver detoxification systems shows direct links to how organisms respond to stress and metabolic disturbances. Detoxification activity has been found to correlate with glucose metabolism as well as variations in water and food intake and diuresis.

Research findings indicate that individual liver function states can affect both diabetes progression and the severity of metabolic complications that accompany this condition. This research explores how biochemical markers in diabetic rats with alloxan-induced diabetes vary according to their liver detoxification activity. Our research aims to increase knowledge about metabolic variability while exploring its impact on diabetes treatment to develop personalized therapeutic strategies.

The study implemented a laboratory-based experimental preclinical approach which used rats to study the metabolic effects related to differences in liver detoxification rates. The research involved 100 sexually mature male white outbred laboratory rats which followed the Guide for the Care and Use of Laboratory Animals: Eighth Edition, National Research Council. The Ethics Committee of the Ministry of Health of Uzbekistan authorized this research through Permit No. 7/26-1953 dated October 18, 2024. The laboratory environment consisted of unrestricted food access alongside water availability and natural light exposure within an air temperature zone of 22±3 °C and air humidity between 30–70%.

We tested the metabolic activity of the CYP450-dependent monooxygenase enzyme system by implementing an in vivo phenotyping approach (Fig. 1). A 40 mg/kg body weight dose of nembutal (pentobarbital) was administered to rats through intraperitoneal injection. The duration of narcotic sleep in minutes functioned as the quantitative indicator for metabolic activity. Rats underwent classification into three distinct groups based on their sleep duration measurements. The animals underwent phenotyping metabolic depression prevention through placement in a thermostat set at 26 °C. Results were recorded and analyzed to categorize metabolic phenotypes.

The laboratory used alloxan monohydrate from Sigma (USA) to create diabetes in rats. The animals underwent overnight fasting followed by a single day of 4% ascorbic acid solution administration to protect against severe diabetogenic effects. A single dose of 150 mg/kg alloxan dissolved in 0.4 mL citrate buffer was administered via intraperitoneal injection to induce diabetes. The laboratory procedures took place from 8:00–11:00 AM to reduce the impact of time-dependent changes [9].

Rats provided blood samples through their tail vein both initially and at three different time points including days 7, 14 and 21. A sterile G-24 needle insertion occurred after hyperemia induction through submersion of the tail into hot water (40–50°C) followed by drying the area. The collected blood samples were placed in heparinized tubes before being spun at 3000 rpm for fifteen minutes to obtain plasma [10]. All hemolyzed samples were eliminated from further analysis.

Plasma Glucose and Hormonal Assays: Automatic analyzer Humastar 100 alongside Human reagents from Germany measured glucose amounts. A Mindray MR 96A immunoassay analyzer performed measurements of Rat Insulin and Rat C-Peptide levels through ELISA kits.

Glucose tolerance test (GTT). Blood collection for initial value took place after an overnight fast. The gastric tube delivery of 2 mL/kg 20% glucose solution occurred as a part of the experimental procedure. Blood samples were collected at four different time points which included 30, 60, 90 and 120 minutes after administration. Researchers plotted glucose concentrations against time to create a glucose concentration-time curve then calculated the area under this curve.

Data were statistically processed using Microsoft Excel. The research presents normally distributed variable results as mean ± standard deviation and non-normally distributed data as median with interquartile range. The Shapiro–Wilk test confirmed the normality of data distribution. The research utilized Student’s t-test to evaluate normally distributed variables but applied the Mann–Whitney U test for nonparametric data analysis. The results achieved statistical significance when the p-value reached less than 0.05.

The analysis of the general rat population revealed that the average duration of Nembutal-induced sleep was 216.36±16.67 minutes, with a range from 76 to 418 minutes. Based on the results of Nembutal-induced sleep, the histogram (Figure 2a) allowed the population to be divided into three distinct groups: rapid metabolizers (duration: 76 to 98 minutes, mean: 91.11±2.35 min), moderate metabolizers (duration: 110 to 150 minutes, mean: 130.29±3.80 min), and slow metabolizers (duration: 183 to 418 minutes, mean: 313.54±15.10 min) (Figure 2b). The differences between these groups were statistically significant (p<0.001, Figure 3). The distribution of metabolizer types in the overall population was 19% rapid, 30% moderate, and 51% slow metabolizers.

The study identified significant differences in carbohydrate metabolism indicators among rats with varying hepatic detoxification activities induced with alloxan diabetes.

In rapid metabolizers, blood glucose levels increased by 2.43-, 2.79-, and 3.66-fold on days 7, 14, and 21 after alloxan injection, respectively, compared to baseline. These increases were statistically significant (P<0.05) (Table 1). However, the difference between days 7 and 14 (1.15-fold) was not statistically significant (P>0.05), whereas the increase from day 7 to day 21 (1.51-fold) was significant (P<0.05). Additionally, the difference between days 14 and 21 (1.31-fold) was also statistically significant (P<0.05).

In moderate metabolizers, blood glucose levels rose by 2.30-, 2.60-, and 2.98-fold on days 7, 14, and 21, respectively. All increases were statistically significant (P<0.05) (Table 1). The change from day 7 to day 14 (1.13-fold) was not statistically significant, whereas the increase from day 7 to day 21 (1.30-fold) reached statistical significance. The difference between days 14 and 21 (1.15-fold) did not reach statistical significance (P>0.05).

In slow metabolizers, blood glucose levels increased by 2.14-, 2.31-, and 2.59-fold at the same time points. These increases were also statistically significant (P<0.05) (Table 1). The change from day 7 to day 14 (1.08-fold) was not significant, while increases between day 7 and day 21 (1.21-fold), and between days 14 and 21 (1.12-fold), were statistically significant (P<0.05).

The results indicate that under alloxan diabetes, rapidly metabolizing animals have significantly higher blood glucose levels compared to moderate and slow metabolizers.

Analysis of insulin levels revealed a progressive decrease in all groups. In rapid metabolizers, insulin levels decreased by 2.21-, 3.00-, and 3.60-fold on days 7, 14, and 21, respectively, compared to baseline (Table 1). In moderate metabolizers, insulin levels dropped by 1.68-, 2.04-, and 2.45-fold over the same time period. In slow metabolizers, the decline was 1.77-, 2.24-, and 2.69-fold, respectively.

In rapid metabolizers, the C-peptide level on day 7 decreased 1.32-fold from baseline, though this reduction was not statistically significant (P>0.05). By days 14 and 21, the C-peptide level was significantly lower — by 2.10- and 2.53-fold, respectively (P<0.05) (Table 1).

Moderate metabolizers exhibited a 1.17-fold reduction in C-peptide on day 7, which was also not statistically significant (P>0.05). However, significant reductions were observed on days 14 and 21, by 1.41- and 1.69-fold, respectively (P<0.05).

In contrast, in slow metabolizers, the decrease in C-peptide levels by 1.17- and 1.47-fold on days 7 and 14, respectively, was not statistically significant (P>0.05). A significant reduction (1.77-fold from baseline) was observed only on day 21 (P<0.05).

These findings demonstrate that rapid metabolizers exhibit a more profound suppression of C-peptide levels compared to moderate and slow metabolizers.

Glucose tolerance tests conducted before the experiment and on day 21 revealed differences among the rat phenotypes. After glucose loading, blood glucose levels in rapidly metabolizing rats increased 2.1-fold from initial value within 60 minutes, whereas moderate and slow metabolizers showed increases of 1.7-fold and 1.5-fold, respectively. These differences between the groups were statistically significant (Figure 4).

To quantify these observations, the area under the glucose concentration–time curve (AUC) was calculated. AUC values were 28.1% and 8.0% higher in rapid and moderate metabolizers, respectively, compared to slow metabolizers.

By day 21, glucose tolerance tests indicated reduced insulin sensitivity in all experimental animals. This was observed in rapid (Figure 5a), moderate (Figure 5b), and slow metabolizers (Figure 5c).

The area under the glucose concentration–time curve increased by 51.1%, 50.6%, and 48.5% from in rapid, moderate, and slow metabolizers, respectively, by day 21 (Figure 6).

The study shows that the capability of rats to detoxify has a major influence on the development of diabetes when exposed to alloxan. The post-Nembutal sleep duration classification system led to the identification of an unexpected high slow metabolizer phenotype rate (51%) which justifies further research on this phenomenon. Blood glucose levels increased more than 270% in rapid metabolizers who also showed the greatest decline in insulin/C-peptide. The present study supports clinical evidence showing CYP450 hepatic enzyme activity affects β-cell resistance to metabolic stress [11][12]. and further proposes that mitochondrial adaptation functions as a new mechanism.

The most severe diabetes development among rapid metabolizers stems from their distinct metabolic response to high glucose conditions. Rapid metabolizers process detoxification well because of their high CYP450 activity but this capability makes them more prone to mitochondrial stress. The mitochondrial activity increases rapidly in rapid metabolizers when they experience high glucose levels which results in excessive ATP and reactive oxygen species (ROS) production [13]. Rivera Nives et al. (2024) demonstrated that the early overactivation of oxidative phosphorylation (OXPHOS) leads to ROS accumulation and electron transport chain inhibition and subsequent glucose unresponsiveness and β-cell death. Rapid metabolizers experience enhanced metabolic flux that drives their severe β-cell failure according to our model [14].

Phenoconversion represents a key process which causes inflammation to reduce CYP450 enzyme activity through mechanisms that do not rely on genetic inheritance. ROS produced by damaged mitochondria trigger the release of proinflammatory cytokines which then decrease the activity of essential detoxification enzymes including CYP3A4 and CYP2C9 [11][12]. The following self-reinforcing metabolic cycle develops because of this sequence: rapid metabolizers → mitochondrial stress and ROS → inflammation → suppression of CYP450 activity → impaired detoxification → accumulation of toxic intermediates → further mitochondrial stress. The research demonstrates this metabolic cycle through rapid metabolizer groups which demonstrate the most severe metabolic and hormonal degradation and thus appear trapped in this feedback loop worse than other groups.

This research stands out because of its extensive phenotypic assessment which shows different carbohydrate metabolism traits between study groups. The alloxan-induced diabetes model had limitations for replicating type 2 diabetes complexity, yet produced consistent metabolic disturbances that allowed for longitudinal study. A key limitation of the present study is the lack of genetic-level information, such as the expression of detoxification-related CYP genes. Although the functional phenotyping approach was sufficient for the aims of this experiment, integrating molecular analyses would considerably strengthen mechanistic interpretation.

Future investigations should validate these findings through studies utilizing streptozotocin-induced type 2 diabetes models, integrating molecular analyses and human population studies. Research into how inflammation together with mitochondrial dysfunction affects CYP450 activity may lead to new personalized therapeutic approaches for metabolic high-risk groups. A combination of mitochondrial-targeted antioxidants with anti-inflammatory agents presents a potential therapeutic approach to protect β-cell function in rapid metabolizers. The research contributes to detoxification system knowledge in diabetes while demonstrating the need for personalized intervention approaches based on individual metabolic characteristics for effective clinical implementation.

This study highlights the significant impact of detoxification capacities on the progression of carbohydrate metabolism disturbances under alloxan diabetes. Rapid metabolizers exhibited the most pronounced increases in blood glucose levels and reductions in insulin and C-peptide levels, indicating a heightened metabolic disruption compared to moderate and slow metabolizers. These findings emphasize the role of individual metabolic phenotypes in influencing the severity of diabetes-induced metabolic changes. Understanding these differences could guide the development of phenotype-specific therapeutic approaches to improve diabetes management and outcomes.

Funding. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest. The authors have no conflict of interest to declare.

Author contributions. All authors contributed to the design of the study, participated in the interpretation of the data, and drafting of the manuscript. All authors reviewed and approved the final, submitted version.