Soon-Tae Kwon

Diosgenin stimulates osteogenic activity by increasing bone matrix protein synthesis and bone-specific transcription factor Runx2 in osteoblastic MC3T3-E1 cells☆

Diosgenin, a steroid saponin extracted from the root of wild yam (Dioscorea villossa) is claimed to have osteogenic property. However, detailed studies providing evidence to this claim have not been fully undertaken. In this study, we investigated the effect of diosgenin on the osteogenesis of murine MC3T3-E1 osteoblastic cells. Cells were cultured with varying levels of diosgenin (0-10 μM) within 25 days of bone formation period. Diosgenin was found to stimulate proliferation within the range of 0.01-5 μM using MTT assay. The medium and cellular levels of Type 1 collagen and alkaline phosphatase (ALP), both of which are major bone matrix proteins, increased within the low range of diosgenin concentration (>0-3 μM), and this pattern was further confirmed by collagen and ALP staining of the extracellular matrix (ECM). The cellular protein expression of ALP and collagen Type 1 was also increased at 0.1-1 μM diosgenin treatment as analyzed by Western blot. Calcium deposition within the ECM also showed the same pattern as assessed by Alizarin Red S and Von Kossa staining. Bone-specific transcription factor runt-related transcription factor 2 (Runx2) and Runx2-regulated osteopontin protein expressions were induced at low concentration (0.1-1 μM) and again decreased with high diosgenin concentrations. Based on our findings, our study suggests that diosgenin can enhance bone formation by stimulating the synthesis and secretion of Type 1 collagen and ALP and bone marker proteins Runx2 and osteopontin expression. The increased levels of these marker proteins, in turn, can increase the formation of calcium deposits within the ECM thereby increasing bone formation.

Marginal zinc deficiency in rats decreases leptin expression independently of food intake and corticotrophin-releasing hormone in relation to food intake.

Zn deficiency reduces food intake and growth rate in rodents. To determine the relationship between Zn deficiency and the regulation of food intake, we evaluated leptin gene expression in epididymal white adipose tissue (eWAT), and hypothalamic corticotropin-releasing hormone (hCRH) and hypothalamic neuropeptide Y (hNPY) of rats Zn-deficient only to show reduced food intake and growth rate but not food intake cycling. Growing male Sprague-Dawley rats (240 g) were randomly assigned to one of four dietary groups: Zn-adequate (ZA; 30 mg/kg diet), Zn-deficient (ZD; 3 mg/kg diet), pair-fed with ZD (PF; 30 mg/kg diet) and Zn-sufficient (ZS; 50 mg/kg diet) (n 8), and were fed for 3 weeks. Food intake and body weight were measured, as were blood mononuclear cells and pancreas Zn levels. eWAT leptin, hCRH and hNPY mRNA levels were determined. Food intake was decreased by about 10 % in ZD and PF rats compared to ZA and ZS rats. Growth and eWAT leptin mRNA levels were unaffected in PF rats but were significantly (P < 0.05) decreased in ZD rats. However, hNPY showed a tendency to increase, and hCRH significantly (P < 0.05) decreased, in both ZD and PF rats. These results suggest that while leptin gene expression may be directly affected by Zn, hNPY and hCRH are likely responding to reduced food intake caused by Zn deficiency.

Leptin Gene Expression and Serum Leptin Levels in Zinc Deficiency: Implications for Appetite Regulation in Rats

Zinc deficiency in animals causes impaired growth and anorexia, and the mechanisms for these symptoms of zinc deficiency are not yet clear. We investigated whether circulating leptin levels and gene expression would be dysregulated under zinc deficiency and what would be the implications for appetite in rats. In study 1, 24 Sprague-Dawley rats were provided consecutively with three different dietary zinc intake levels: Zn-adequate (30 mg/kg of diet), Zn-depleted (1 mg/kg of diet), and Zn-replete (50 mg/kg of diet), for 1, 2, and 2 weeks, respectively. At the end of each dietary period, one-third of the rats were killed. In study 2, rats were assigned to one of the four Zn diet groups: Zn-adequate (30 mg/kg of diet), pair-fed (30 mg/kg of diet), Zn-deficient (1 mg/kg of diet), or Zn-sufficient (50 mg/kg of diet), and were fed for 4 weeks. Tissue Zn and serum leptin were measured, and leptin gene expression in adipose tissues (inguinal and abdominal) was determined by reverse transcription-polymerase chain reaction and northern blotting. Blood subfractions as plasma, red blood cells, and mononuclear cells and liver Zn level were decreased during the Zn-depletion period (P <.05). Serum leptin showed a tendency to increase during the Zn-depletion period and decreased back to the level of the Zn-repletion period. Leptin mRNA levels in inguinal adipocytes also increased during the Zn-depletion (P <.05) and Zn-deficient periods, which is consistent with the change in serum leptin. However, the decrease in leptin mRNA in abdominal adipocytes was not consistent with the increase in inguinal leptin levels and the change in serum leptin. Increased leptin levels in linguinal adipocytes is consistent with the expected physiological change of a decrease in appetite under Zn deficiency. However, before coming to any firm conclusion, further studies on adipose tissue-specific leptin expression, including the appetite-related neuropeptides, are necessary for clarifying the cause of lower appetite in zinc deficiency.

Cloning of Cytochrome P450 Gene involved in the Pathway of Capsidiol Biosynthesis in Red Pepper Cells

In order to measure the enzyme activity of 5-epi-aristolochene hydroxylase, one of cytochrome P450 (P450) enzymes in eicitor-treated pepper cell, we used in vivo assay method and demonstrated a dramatic suppression of the activity by P450-inhibitors, ancymidol and ketocornazole. Using RT-PCR method with degenerate primer of the well conserved domains found within most P450-enzymes, and using cDNA library screening method, one distinct cDNA, being designated P450Hy01, was successfully isolated from elicitor-treated pepper cells. P450Hy01 mRNA was all induced in elicitor-treated cells whereas never induced in control cells. Moreover, levels of P450Hy01 expression were highly correlated with the levels of extracellular capsidiol production by different elicitors in cell cultures. P450Hy01 transcript was also induced by several other elicitors such as, cellulase, arachidonic acid, jasmonic acid, yeast extract as well as UV stress. P450Hy01 sequence contained high probability amino acid matches to known Plant P450 genes and ORF with a conserved FxxGxRxCxG heme-binding domain. P450Hy01 cDNA showed 98% of homology in sequence of nucleotide as well as amino acid to 5-epi-aristolochene-1, 3-hydroxylase (5EAl, 3H) which has been isolated in tobacco cells, suggesting that P450Hy01 is prominent candidate gene for P450-enzyme encoding 5EAl, 3H in pepper cell.