This study tested LIM on 3T3-L1 adipocytes and high-calorie diet-induced obese rats for its anti-obesity effect on the 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway, as well as its optimally effective dose.

In the past few years, obesity has become one of the most significant clinical problems worldwide. It results from an imbalance between energy intake and energy expenditure, leading to an accumulation of excessive body fat in the body.

Approximately 760 million people worldwide are obese as of 2020, and it is expected that 1 billion adults will be obese by 2030. There is no doubt that obesity contributes to metabolic disorders such as hypertension, type 2 diabetes, hyperlipidemia, and cardiovascular disease.

Monoterpene D-limonene (LIM) is primarily found in citrus essential oils. D-limonene, a volatile monoterpene compound produced by plants, has been shown to inhibit adipocyte differentiation and stimulate lipolysis, making them possible anti-obesity agents.

Besides citrus essential oils, LIM is also found in citrus pulp, the main source of human D-limonene intake. LIM is primarily found in citruses, such as lemon, orange, and grapefruit. 

Several biological activities have been evaluated using LIM, such as anti-oxidation, anti-inflammation, and blood pressure regulation. In addition, LIM has demonstrated low toxicity and no evidence of risk in animal studies. Therefore scientists can use it to evaluate various biological activities.

Methodology and Results

The experts determined oil red O and triglyceride (TG) content ten days after treatment with 0.05–0.4 mg/mL of LIM in 3T3-L1 adipocytes. The results showed that more than 0.05 mg/mL of LIM inhibited lipid accumulation by decreasing oil red O in 3T3-L1 adipocytes.

3T3-L1 adipocytes receiving 0.2 mL and 0.4 mL LIM also showed decreased TG levels, whereas Wistar rats received 154 mg/kg LLIM and 1000 mg/kg HLIM.

Based on the results, LLIM and HLIM were associated with decreased body weight, total fat tissue weight, and serum low-density lipoprotein cholesterol (LDLc). In addition, HLIM was associated with lowered serum TG, a greater increase in serum lipase, and lower levels of HDLc.

The results also showed LIM to activate the AMPK signaling pathway in obesity-induced 3T3-L1 adipocytes and obese rats after high-calorie diets.

The researchers found that activating AMPK inhibits obesity by regulating mRNA expression of adipogenesis, lipogenesis, and lipolysis.

In 3T3-L1 adipocytes and rats with high-calorie diet-induced obesity, LIM is shown to regulate the AMPK signaling pathway, demonstrating its anti-obesity properties. According to the dose-response relationship, LIM (154 mg/kg) would be the optimal dose for reducing obesity caused by high-calorie consumption.

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