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Involvement of sphingomyelinase in insulin-induced phosphatidylinositol 3-kinase activation¹

CHUANSHU HUANG^*,, WEI-YA MA, MIN DING^*, JINGXIA LI^*, XIANGLIN SHI^*, VINCENT CASTRANOVA^*, VAL VALLYATHAN^*, ANN M. BODE and ZIGANG DONG²

^* The Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia, USA; and
The Hormel Institute, University of Minnesota, Austin, Minnesota, USA

²Correspondence: The Hormel Institute, University of Minnesota, 801 16th Ave. N.E., Austin, MN 55912, USA. E-mail: zgdong{at}smig.net

SPECIFIC AIM

Thismanuscript aims to elucidate the mechanism by which insulin activates phosphatidylinositol 3-kinase (PI-3 kinase), especially the role of sphingomyelinase (SMase) in insulin-induced PI-3 kinase activation.

PRINCIPAL FINDINGS

1. Induction of PI-3 kinase activation by insulin, C2-ceramide, or PCho in mouse epidermal JB6 cells
To determine the possible role of SMase and its products in PI-3 kinase activation, we exposed JB6 cells to either C2-ceramide or PCho. Both C2-ceramide and PCho induced strong activation of PI-3 kinase in JB6 cells. The induction of PI-3 kinase activity by C2-ceramide or PCho was blocked by either pretreatment of cells with wortmannin or by overexpression of the dominant negative mutant of PI-3 kinase (P85).

2. Insulin induces PI-3 kinase activation in normal JY lymphoblasts, but not in SMase-deficient MS1418 lymphoblasts
To investigate the role of SMase in insulin-induced PI-3 kinase activation, we exposed both EBV-transformed normal lymphoblasts (JY) and SMase-deficient lymphoblasts (MS1418) to insulin for PI-3 kinase induction. The results show that insulin-induced PI-3 kinase was observed only in normal JY cells, but not in SMase-deficient MS1418 cells (Fig. 1 ). These results suggest that SMase is involved in PI-3 kinase activation by insulin.

View larger version (25K): [in this window] [in a new window]   Figure 1. Induction of PI-3 kinase by insulin in normal lymphoblasts, but not in SMase-deficient MS1418 lymphoblasts. Lymphoblasts (JY or MS1418) were or were not treated with insulin for 10 min at 37°C in 5% CO2. The cells were then harvested, and PI-3 kinase activity (PIP3) was measured as described in Materials and Methods.

3. Restoration of PI-3 kinase activation by C2-ceramide, PCho, or SMase in SMase-deficient MS1418 cells
To demonstrate a role for SMase in PI-3 kinase activation further, we treated MS1418 cells with C2-ceramide, PCho, or SMase to examine whether the deficiency of PI-3 kinase activation in MS1418 cells could be restored. Adding exogenous C2-ceramide directly into the cell culture medium caused activation of PI-3 kinase in both JY and MS1418 cells. The time-course study indicated that exposure of MS1418 cells to SMase, PCho, or C2-ceramide induced a very strong activation of PI-3 kinase within 5 min (Fig. 2 ) and the activity returned toward the basal level by 15 min after exposure (Fig. 2) . The results from a concentration response study are consistent with the above findings that treatment of cells with SMase can restore PI-3 kinase activation in MS1418 cells. These experiments provide evidence that the lack of insulin-induced PI-3 kinase activation in MS1418 cells is due to a deficiency of SMase.

View larger version (46K): [in this window] [in a new window]   Figure 2. Restoration of PI-3 kinase activation in MS1418 cells by C2-ceramide, PCho, or Smase. MS1418 cells were exposed to SMase (0.5 U/ml), Pcho (20 mM), or C2-ceramide (20 µM) for the time as indicated. The cells were then harvested and PI-3 kinase activity (PIP3) was measured as described in Materials and Methods.

CONCLUSIONS AND SIGNIFICANCE

Our present studies demonstrate that SMase is involved in insulin-induced activation of PI-3 kinase in mouse epidermal JB6 cells, human lymphoblast (JY), and MS1418 cells. This conclusion is based on the following experimental observations: 1) insulin, C2-ceramide, or phosphocholine (PCho) can stimulate PI-3 kinase activity in JB6 cells; 2) this insulin-induced activation of PI-3 kinase is observed in the normal human lymphoblast cell line, JY, but not in a SMase-deficient cell line, MS1418; and 3) the deficiency of PI-3 kinase activation in MS1418 cells could be restored by exposure of the cells to SMase, C2-ceramide, or PCho.

Numerous extracellular stimulations can result in activation of SMase. This result, in turn, causes hydrolysis of sphingomyelin, a phospholipid largely confined to the outer leaflet of cellular membranes, and stimulates the generation of ceramide and PCho. These activating agents include UV or ionizing irradiation, heat shock, nerve-growth factor, TNF-, endotoxin, interferon-, interleukin-1, Fas, and CD28. Also, a number of direct targets for ceramide action have been identified, which include ceramide-activated protein kinase, ceramide-activated protein phosphatase, and protein kinase C. Growing evidence indicates an important role of SMase and its products, ceramide, and PCho as secondary messengers in the regulation of cell growth and differentiation, cell–cell contact, and oncogenesis, depending on cell type.

Our previous studies also demonstrated that UV-induced JNKs activation depends on SMase. In the present study, we found that SMase, ceramide, and PCho induce strong activation of PI-3 kinase in JB6 cells. In the lymphoblast cell system, insulin-induced PI-3 kinase activation is observed only in a normal human lymphoblast (JY) but not in a SMase-deficient cell line (MS1418). Moreover, exposure of cells to SMase, ceramide, or PCho leads to significant activation of PI-3 kinase in both JY cells and MS1418 cells. Exogenous ceramide, PCho, or SMase could restore PI-3 kinase activation in SMase-deficient cells, which suggests that the lack of response of MS1418 cells to insulin in terms of PI-3 kinase activation is due to its deficiency of SMase. Thus, we conclude that SMase and its products are involved in insulin-induced PI-3 kinase activation.

Because PI-3 kinase plays an important role in mediating metabolism of lipids, we speculate that deficiency of PI-3 kinase activation in response to insulin may at least be part of the molecular mechanism for abnormal metabolism of lipids in patients with Niemann-Pick disease.

In conclusion, we used several approaches to study the role of SMase in insulin-induced PI-3 kinase activation (Fig. 3 ). PI-3 kinase activation can be observed in JB6 cells treated with insulin, ceramide, or PCho. Insulin induces PI-3 kinase activation in normal lymphoblasts but not in SMase-deficient lymphoblasts. Exposure of SMase-deficient cells to SMase, ceramide, or PCho can restore PI-3 kinase activation. These results demonstrate that SMase plays a role in insulin-induced PI-3 kinase activation. These data reveal that the lack of PI-3 kinase activation by insulin may be a possible mechanism for abnormal lipid metabolism in Niemann-Pick disease. Further investigations will focus on the effects of SMase, ceramide, or PCho on uptake and esterification of low-density lipoprotein-derived cholesterol in animals and cultured cells.

View larger version (31K): [in this window] [in a new window]   Figure 3. Involvement of sphingomyelinase in insulin-induced PI-3 kinase activation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0520fje ; to cite this article, use (February 5, 2001) FASEB J. 10.1096/fj.00-0520fje

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This Article Full Text (PDF) All Versions of this Article: 15/6/1113 00-0520fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by HUANG, C. Articles by DONG, Z. Search for Related Content PubMed PubMed Citation Articles by HUANG, C. Articles by DONG, Z.