تأثیر 21 هفته HIIT و مکمل‌‌دهی ال‌‌کارنیتین بر بیان ژن برخی عوامل مرتبط با تغییر بافت چربی سفید به قهوه‌‌ای و پروتئین جفت نشده نوع-1 در رت‌‌های نر چاق

Extended abstract 
Background and Aim: Obesity is a risk factor for metabolic and cardiovascular diseases. White adipose tissue (WAT) stores excess fat, preventing its accumulation in vital organs; however, excessive WAT accumulation can contribute to obesity and a reduced in brown adipose tissue (BAT) activity. MicroRNAs, particularly miR-455, play a critical role in regulating the differentiation of brown adipocytes, a process that counteracts obesity. MiR-455 enhances the differentiation of brown fat cells by targeting key regulators of lipogenesis and inhibiting hypoxia-inducible factor 1 alpha (HIF-1α). Inhibition of HIF-1α activates Adenosine monophosphate-activated protein kinase-1 alpha (AMPK-1α), leading to increased expression of Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and Peroxisome proliferator-activated receptor-gamma (PPAR-γ), ultimately boosting mitochondrial biogenesis and thermogenesis in WAT. High-intensity interval training (HIIT) and L-carnitine supplementation are known as stimulants for converting WAT phenotype to beige and activating BAT; however, their impact on the UCP1/HIF-1α/miR-455 axis remains unclear. The present study aims to investigate the effects of 12 weeks of HIIT and L-carnitine supplementation on the expression of this axis in obese male rats, paving the way for innovative treatments for obesity and metabolic disorders.
Materials and Methods: Thirty-six male rats were used in this experimental study. Obesity was induced by eight-week of a high-fat diet (60% fat, 20% carbohydrate, 20% protein), leading to an approximate weight of 450 g. After induction, the rats were fed a standard diet ad libitum. The rats were then randomly assigned to one of four groups: control (C), Training (T), supplementation (S), and supplementation+Training (ST). Following the induction period, all rats were fed standard chow. To determine maximal running speed, groups T and ST underwent a Bedford treadmill test. This test consisted of ten 3-min stages, beginning at 5 meters per min on a 25-degree incline, with speed increments of 5 meters per stage until exhaustion. A 12-week HIIT protocol (5 days/week) comprised: 5-min warm-up and cool-down (<60% max speed), followed by ten 4-min intervals at 85-90% max speed, with 2-min active recovery (50-60% max speed) between intervals. To account for potential changes in maximal speed and prevent uniform training intensity, a retest of maximal speed was performed every two weeks on a non-training day for groups T and ST. Groups S and ST received daily oral L-carnitine (30 mg/kg body weight) by gavage one hour after training; groups C and T received an equivalent volume of distilled water. Forty-eight hours post-training and following an overnight fast, rats were euthanized. Subcutaneous inguinal adipose tissue samples were collected and processed for analysis. The expression levels of miR-455, HIF-1α, and UCP1 were determined using RT-qPCR, Western blotting, and immunofluorescence, respectively. After confirming data normality (Shapiro-Wilk test) and homogeneity of variances (Levene test), within-group comparisons were performed using paired t-tests. Between-group comparisons were performed using one-way ANOVA, with post hoc analysis using the Bonferroni correction. Statistical analyses were performed using SPSS version 26  at p<0.05 level. Graphs were generated using Excel 2007.
Findings: The results of the Shapiro-Wilk test showed that the weight distribution of all rats was normal at the beginning of the study (p=0.13). A dependent t-test confirmed the weight gain of all rats at the end of the eight-week high-fat diet (p=0.001). After dividing the rats into four groups and implementing the respective interventions, a significant difference was observed between the weights of the groups (p=0.001), such that groups T and ST had a significant decrease compared to group C (p=0.003 and p=0.001 respectively) and compared to group S (p=0.02 and p=0.001 respectively), but no significant changes were observed between groups ST and T, as well as groups S and C (p=0.001). Finally, group S had lower weight compared to group C and group ST had lower weight compared to T.
Regarding the expression of miR-455, a significant difference was observed between the groups (p=0.001). Group T had a significant decrease (p=0.001) and group S had a significant increase (p=0.001) compared to group C. Group ST did not have a significant increase compared to group C (p=0.07). The difference between other pair of groups was significant (p=0.001)(Figure 1).
The results for HIF-1α expression indicated a significant difference between the groups (p=0.001), such that groups T, S and ST had a significant increase compared to group C (p=0.001). There was no significant difference between groups T and S (p=1.00)( Figure 1).
A significant difference was also observed in the expression of UCP1 protein (p=0.001), and all groups had a significant increase compared to group C (p=0.001), but there was no significant difference between groups T and ST (p=0.08)( Figure 1).
The study of adipose tissue images showed that group C had large white fat cells with fewer mitochondria and lower capillary density, indicating low metabolic activity and inflammation. Group T had a higher number of small brown/beige multilocular fat cells and higher capillary and mitochondrial density, indicating higher metabolic activity. Group S showed slight improvement compared to group C, while group ST showed significant improvement in metabolic parameters, although not to the same extent as group T . These findings were consistent with the observed weight changes in each group.
 
 
 
 
Figure 1. Comparison of mir-455 (μg/mL), HIF-1α (μg/mL) and UCP1 (μm) between groups. *significant difference between groups at p<0.05.
Conclusion: This research examined the effects of L-carnitine and HIIT on factors influencing WAT browning. L-carnitine supplementation alone elevated levels of miR-455, HIF-1α, and UCP1, yet exerted no discernible impact on body weight. In contrast, HIIT paradoxically increased UCP1 activity while reducing miR-455 expression and stimulating alternative pathways associated with WAT-to-BAT conversion; this, coupled with the caloric expenditure inherent in training, resulted in weight loss. The combined administration of L-carnitine and HIIT synergistically activated UCP1 and augmented caloric consumption. These findings offer potential avenues for combating obesity and provide promising evidence for promoting WAT browning. As a novel study, this research suggests that the concurrent administration of HIIT and L-carnitine may effectively modulate BAT activity, however, further research is needed to confirm these observations.
Ethical Considerations: This study received ethical approval from the Biomedical Ethics Committee of Tabriz University (IR.TABRIZU.REC.1401.042). The research was conducted using the minimum number of rats necessary and adhered to optimal laboratory animal conditions (12:12 light-dark cycle, 50% ± 3% relative humidity, and 22±2°C temperature). Non-invasive and low-pain methods, minimizing distress, were employed for stimulating the rats. All researchers completed mandatory training courses on animal handling and ethical principles.
Compliance with ethical guideline: Alternative methods to animal experimentation were considered whenever feasible, and laboratory procedures were designed to minimize pain and suffering.
Funding: All costs associated with this research, including analyses, materials, and publication fees, were covered by the authors. No external funding was received from any institution or organization.
Conflict of interest: The authors declare no conflicts of interest pertaining to this research.