The mechanisms for the insulin resistance induced by hyperglycemia were investigated by studying the effect of high glucose concentration (HG) and its modulation by thiazolidine derivatives, on insulin signaling using Rat 1 fibroblasts expressing human insulin receptors (HIRc). Incubating HIRc cells in 27 mM D-glucose for 4 days impaired the insulin-stimulated phosphorylation of pp185 and receptor beta-subunits. Both protein kinase C activities and phorbol dibutyrate binding to intact cells were unchanged; however, cytosolic protein-tyrosine phosphatase (PTPase) activity increased within 1 h prior to the impairment of insulin receptor kinase in HG cells (Maegawa, H., Tachikawa-Ide, R., Ugi, S., Iwanishi, M., Egawa, K., Kikkawa, R., Shigeta, Y., and Kashiwagi, A. (1993) Biochem. Biophys. Res. Commun. 197, 1078-1082). Increased PTPase activity was consistent with a 2-fold increase in the amount of PTP1B, and anti-PTP1B antibody inhibited this increment of cytosolic PTPase activity in HG cells. Co-incubating cells with pioglitazone prevented these abnormalities in cytosolic PTPase, the PTP1B content and the impaired phosphorylation of pp185 and receptor beta subunits in HG cells. Finally, HG cells had impaired insulin-stimulated alpha-amino-isobutyric acid uptake, which was ameliorated by exposure to thiazolidine derivatives. In conclusion, exposing cells to high glucose levels desensitizes insulin receptor function, and thiazolidine derivatives can reverse the process via the normalization of cytosolic PTPase, but not of protein kinase C.

Endothelin (ET), a novel vasoconstrictor peptide containing three isopeptides [ET-1, ET-2, and ET-3 (ETs)], has various biological effects including vasoconstriction, mitogenesis, and steroidogenesis. We examined the ET-1-like immunoreactivity level in porcine follicular fluid and culture medium of porcine granulosa cells by RIA. The ET level in the follicular fluid was 9.4-14.2 pg/ml. These levels were within 0.42 to 0.62-fold of the porcine plasma level (22.7 +/- 3.1 pg/ml) (mean +/- SE). ET was detected in the culture medium of granulosa cells with and without LH treatment at the concentration of 56 +/- 9.3 and 4.9 +/- 1.2 pg/10(6) cells.h, respectively. We also examined whether ETs affect the luteinization of granulosa cells. ETs inhibited the LH-stimulated progesterone and cAMP accumulation in cultured porcine granulosa cells in a dose-dependent manner with an EC50 of 5 x 10(-11) M. ET-1, ET-2, and ET-3 (5 x 10(-8)M inhibited progesterone accumulation by 62.3 +/- 1.8, 59.8 +/- 4.0, and 63.3 +/- 5.7% in 6-day cultures, respectively, and significant inhibition was observed within 24 h of culture. ET-1, ET-2, and ET-3 (5 x 10(-8) M) inhibited the LH-stimulated cAMP accumulation in granulosa cells by 54.8 +/- 2.3, 55.4 +/- 7.1, and 55.5 +/- 6.2%, respectively, whereas they did not affect basal cAMP levels. As well as progesterone accumulation, ETs partially inhibited LH-stimulated morphological transformation of granulosa cells. In this study, we demonstrated that ET exists in follicular fluid and in the culture medium of granulosa cells, and that ET inhibited LH-induced progesterone accumulation, morphological transformation, and cAMP accumulation in cultured porcine granulosa cells. These findings suggest that ET acts as a modulator of steroid metabolism in preovulatory follicles.

To elucidate the roles of SHP-2, we generated transgenic (Tg) mice expressing a dominant negative mutant lacking protein tyrosine phosphatase domain (⌬PTP). On examining two lines of Tg mice identified by Southern blot, the transgene product was expressed in skeletal muscle, liver, and adipose tissues, and insulin-induced association of insulin receptor substrate 1 with endogenous SHP-2 was inhibited, confirming that ⌬PTP has a dominant negative property. The intraperitoneal glucose loading test demonstrated an increase in blood glucose levels in Tg mice. Plasma insulin levels in Tg mice after 4 h fasting were 3 times greater with comparable blood glucose levels. To estimate insulin sensitivity by a constant glucose, insulin, and somatostatin infusion, steady state blood glucose levels were higher, suggesting the presence of insulin resistance. Furthermore, we observed the impairment of insulin-stimulated glucose uptake in muscle and adipocytes in the presence of physiological concentrations of insulin. Moreover, tyrosine phosphorylation of insulin receptor substrate-1 and stimulation of phosphatidylinositol 3-kinase and Akt kinase activities by insulin were attenuated in muscle and liver. These results indicate that the inhibition of endogenous SHP-2 function by the overexpression of a dominant negative mutant may lead to impaired insulin sensitivity of glucose metabolism, and thus SHP-2 may function to modulate insulin signaling in target tissues. SHP-2 (also referred to as PTP1D, PTP2C, SHPTP2, or SYP)is a ubiquitously expressed protein-tyrosine phosphatase (PTPase) 1 containing a single PTPase domain and two adjacent Src homology (SH) 2 domains near its N terminus which specifically associate with a variety of tyrosine-phosphorylated proteins upon growth factor stimulation (1-5). SHP-2 is the mammalian homologue of Drosophila Corkscrew, whose gene product potentiates the Drosophila homologue of mammalian c-raf to positively transmit signals downstream of the Torso receptor tyrosine kinase (6). Furthermore, SHP-2 has been reported to play an important role in mesodermal induction in oocyte by regulation of mitogen-activated protein (MAP) kinase activity (7).Regarding the roles of SHP-2 in tyrosine kinase signaling, several lines of evidence indicate that SHP-2 acts as a positive mediator in growth factor signaling such as that by plateletderived growth factor and epidermal growth factor (4,5,8,9). After stimulation by these ligands, SHP-2 is tyrosine-phosphorylated and bound to Grb2-SOS complex, resulting in activation of p21 ras and MAP kinase cascade. On the other hand, in the case of insulin signaling, SHP-2 is not tyrosine-phosphorylated in response to insulin stimulation. However, insulin induces the association of IRS-1 with SHP-2 (10, 11), and the expression of either a catalytically inactive mutant SHP-2 (Cys/Ser) or a deletion mutant lacking PTPase domain in Chinese hamster ovary cells overexpressing insulin receptors (CHO-IR) results in the attenuation of the insulin-stimulated MAP kinase activity, ...

In this study, the absorption, distribution and excretion of ceramide were examined in rats. After a single oral administration of (3)H-ceramide, mean plasma concentration of radioactivity reached maximum at approximately 10.67 hr and decreased with a T(1/2) of 67.12 hr. The mean cumulative excretion of radioactivity in urine and feces accounted for approximately 4.79% and 87.44%, respectively, of the dose. At 96 hr after dosing, 1.67% and 3.67%, respectively, of the dose were still present in the skin and carcass. The radioactivity in the skin at 12 hr was lower than that in plasma and the ratio of skin to plasma concentration was 0.7. However, at 120 hr after dosing, the ratio of skin to plasma concentration increased to 4. A detailed analysis of the distribution of radioactivity in a section of skin showed that radioactivity was located in the dermis and epidermis. At 168 hr, the radioactivity in the epidermis was 8.0% of the radioactivity in skin. The results of the present study clearly demonstrate that some ceramide orally administered is distributed gradually in the dermis after gastrointestinal absorption, followed by transfer from the dermis to the epidermis.

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