Influence of Tomatine and Tomatidine on Testosterone Secretion from Testicular Leydig Cells

Tomatine and tomatidine are steroidal glycoalkaloid occurred in all parts of tomato plant, especially in green fruit. Previous studies showed that tomatine and tomatidine inhibit fungal infection, lower plasma cholesterol and triglycerides, enhance the immunity, anti-inflammation, and induce cancer cell apoptosis. However, the effect of tomatine and tomatidine on testosterone is not clear. Also, it is still unknown that the signal transduction for tomatine and tomatidine on Leydig cells. Regucalcin (RGN) is a kind of protein for binding of calcium ion. RGN is most abundant in liver and kidney. Also, RGN exists in prostate gland, ovary, breast, testicular Leydig cells, and sperm. Sirtuins belong to a family of NAD+-dependent histone deaceylase. There are 7 kinds of sirtuins (SIRT1~SIRT7) found in human tissues. SIRT1 and SIRT2 exist in cytoplasm and nucleus. Recent study indicated that SIRT1 has been found in testis. In addition, SIRT1 has been demonstrated to be related to the reproductive ability and anti-inflammatory. Therefore, RGN and SIRTs could be important factors for reproduction. Most flavonoids used in research showed the inhibitory effects on immunological or endocrinological system. Tomatidine and arecoline (Am J Physiol Endocrinol Metabol. 2008, 295:E497-E504), as PI’s knowledge, are two substances purified from plants, which showed acute stimulatory effects on testosterone secretion from Leydig cells. The objective of this three-year proposal are: (1) to investigate the effects of tomatine and tomatidine on testosterone secretion, MAPK phosphorylation, the expression of steroidogenesis enzymes, SIRT, and regucalcin (RGN) in TM3 (rat Leydig cell lines) cells; (2) to understand the effects of tomatine and tomatidine on the secretion of testosterone and the expression of signal molecules in rat Leydig cells in vitro; (3) to study the effects of tomatine and tomatidine on plasma testosterone and LH concentration in different ages and periods after oral ingestion. TM3 cells and isolated rat Leydig cells will be cultured in RPMI-1640 and stimulated with different concentrations of tomatine and tomatidine. Different concentrations of tomatine and tomatidine will be ingested by oral to 1, 3, and 6 months old of rats. Tomatine and tomatidine will be ingested by oral to adult rats for 1 or 14 days. Testosterone concentrations in plasma and collected media will be measured by enzyme-linked immunosorbent assay (ELISA). Western blot will be employed for the determination of signal molecules, including SIRT, phosphorylated MAPK (ERK1/2, JNK, and p38), RGN, and the enzymes for steroidogenesis (e.g. StAR, P450scc, etc.). Messenger RNA expression of SIRT, RGN, and the enzymes for steroidogenesis will be analyzed by RT-PCR. Preliminary data showed that tomatidine could stimulate the testosterone secretion from TM3 cells at 6 and 9 hour (Fig. 1). In addition, the existence of SIRT1, SIRT2, and RGN expression in TM3 cells has been verified (Fig. 2). Enhanced testosterone secretion was observed in TM3 cells treated with tomatidine (1~10 μM) for 6 h (Fig. 4). Increased testosterone secretion was found in rat Leydig cells treated with hCG (0.05IU/ml) and tomatine (Fig. 5A) or tomatidine (10-8 ~ 10-6 M) (Fig. 5B). The increased expression of P450c17 enzyme has also been found in TM3 cells treated with 10~50 μM tomatidine for 24 h (Fig. 6). We hope that these studies will provide relevant information for the understanding of influence of tomatine and tomatidine on human health.

Project ID:PB10408-6661
External Project ID:MOST104-2320-B182-012