The main results of this study were that L-Cit reduced appetite in both obese/diabetic KK-Ay mice and high-fat diet fed SD rats. The underlying mechanism was identified as increased expression of POMC in the hypothalamus. There were two signaling pathways, leptin signaling and insulin signaling, which regulated POMC expression.
Leptin is a key regulator of feeding and long-term energy homeostasis that acts on discrete neuronal pathways to reduce food intake and body fat content.11) One mechanism implicated in leptin regulation of cellular function is the activation of JAK2 and STAT3 signaling12,13) which appears to play a major role in the energy homeostasis mechanism. Phosphorylated STAT3 is translocated from the cytoplasm to the nucleus, stimulating POMC expression and inhibiting agouti-related protein (Agrp). In this study we showed that L-Cit does not phosphorylate JAK2 and STAT3, which are involved in leptin signaling. These results suggest that the change in POMC neuronal expression is not involved in leptin signaling in L-Cit treatment.
Akt is a central enzyme in insulin signaling and Akt activity is regulated by various factors, such as IR-IRS1 signaling and mTOR signaling. We clarified that L-Cit increased the ratio of phosphorylated Akt (Ser473) in the hypothalamus of high-fat diet fed SD rats. In our study, L-Cit did not affect IR-IRS1 signaling so we examined Akt-mTOR signaling as another possible pathway. mTOR, a phosphorylation target of Akt, was stimulated and increased expression by amino acids.14) mTOR is the catalytic subunit of complexes mTORC1 and mTORC2. If mTORC2 phosphorylation increases, Akt phosphorylation also increases FoxO1 phosphorylation, which increases POMC expression. On the other hand, mTORC1 activity causes a negative feedback loop on the insulin signaling pathway. Activation of mTORC1 leads to phosphorylation of downstream S6K, which participates in several processes including protein synthesis and proliferation.6,15) In addition, increased activity of the mTOR-S6K signaling pathway leads to serine phosphorylation of IRS1, creating a negative feedback loop to insulin signaling that attenuates insulin sensitivity.16,17) In our study, we considered that L-Cit stimulated mTORC2 phospholyration because phosphorylation of factors involved in signaling through mTORC1, such as S6K and IRS1, did not change between the two groups (Fig. 4).
|Fig. 4. Signaling of Appetite Suppression in the Hypothalamus of L-Cit Treated Animals|
In our previous study using 3T3-L1 adipocytes, we investigated into factors involved in adipose synthesis, lipolysis and metabolism. As a result, every factor did not change by L-Cit treatment, therefore we considered appetite suppression by L-Cit administration was main mechanism of body weight reduction.
In our study, we used two types of obesity model animals for clarifying the effect of L-Cit on metabolic syndrome. First, KK-Ay mice were used as a metabolic phenotype-associated model and we examined food intake level and effects on induction of obesity. As a result, food intake and free fatty acid level decreased significantly in KK-Ay mice despite not significantly decreasing in high-fat diet fed SD rats. High-fat diet fed SD rats were used as a model for a condition near obesity.18) By giving a high-fat diet to SD rats for 11 weeks, body weight increased further so we were able to obtain obesity model rats. Actually, body weight and cholesterol level tended to decrease, and insulin level and HOMA-IR significantly decrease in the L-Cit group in high-fat diet fed SD rats. From the results of reduction of free fatty acid level and serum insulin level in KK-Ay mice and cholesterol level and insulin level in SD rats with L-Cit treatment, we concluded that L-Cit improves lipid metabolism and glucose metabolism in the two types of obesity model animals. In addition, blood glucose level in KK-Ay mice and high-fat fed SD rats also were not significant differences between two groups but serum insulin level significantly decreased in the L-Cit group. Our data suggested that L-Cit reduced insulin level in KK-Ay mice and high-fat diet fed SD rats having nothing to do with blood glucose level. The reason why we got this result was improvement of insulin sensitivity by recovering metabolic syndrome with L-Cit treatment.
We considered why results of the two types of metabolic syndrome model animals differed, and one possibility was the difference of animal species. As results, L-Cit affects metabolic syndrome prophylaxis and improvement in two types of model animal, KK-Ay mice and high-fat diet fed SD rats.
We used high dose L-Cit however ALT and AST levels, which indicated liver function, were not affected. In addition, there was no trouble with condition of bowel movement and activity during our experiment period. Moreover, we observed the stomach and intestines when we dissected the organ, we cannot find abnormal condition. Therefore we considered L-Cit dose in our study did not have toxicity and side effect.
From now on, we will continue with our study; L-Cit may become a new compound for treatment of metabolic syndrome through appetite suppression and be used in clinical fields in the future.