Suppression of PC-1/ENPP-1 expression improves insulin sensitivity in vitro and in vivo.

Plasma cell membrane glycoprotein-1, or ectonucleotide pyrophosphatase/phosphodieterase (PC-1/ENPP1) has been shown to inhibit insulin signaling in cultured cells in vitro and in transgenic mice in vivo when overexpressed. Furthermore, both genetic polymorphism and increased expression of PC-1 have been reported to be associated with type 2 diabetes in humans. Thus it was proposed that PC-1 inhibition represents a potential strategy for the treatment of type 2 diabetes. However, it has not been proven that suppression of PC-1 expression or inhibition of its function will actually improve insulin sensitivity. We show in the current study that transient overexpression of PC-1 inhibits insulin-stimulated insulin receptor tyrosine phosphorylation in HEK293 cells, while knockdown of PC-1 with siRNA significantly increases insulin-stimulated Akt phosphorylation in HuH7 human hepatoma cells. Adenoviral vector expressing a short hairpin RNA against mouse PC-1 (PC-1shRNA) was utilized to efficiently knockdown PC-1 expression in the livers of db/db mice. In comparison with db/db mice treated with a control virus, db/db mice treated with the PC-1shRNA adenovirus had approximately 80% lower hepatic PC-1 mRNA levels, approximately 30% lower ambient fed plasma glucose, approximately 25% lower fasting plasma glucose, and significantly improved oral glucose tolerance. Taken together, these results demonstrate that suppression of PC-1 expression improves insulin sensitivity in vitro and in an animal model of diabetes, supporting the proposition that PC-1 inhibition is a potential therapeutic approach for the treatment of type 2 diabetes.

Evidence that inhibition of insulin receptor signaling activity by PC-1/ENPP1 is dependent on its enzyme activity.

Plasma cell membrane glycoprotein-1 or ectonucleotide pyrophosphatase/phosphodiesterase (PC-1/ENPP1) has been shown to inhibit insulin signaling, and its genetic polymorphism or increased expression is associated with type 2 diabetes in humans. Therefore, PC-1 inhibition represents a potential strategy in treating diabetes. Since patients with phosphodiesterase/pyrophosphatase deficient PC-1 manifest abnormal calcification, enhancing insulin signaling by inhibiting PC-1 for the treatment of diabetes will be feasible only if PC-1 phosphodiesterase/pyrophosphatase activity needs not be significantly diminished. However, whether inhibition of insulin receptor signaling by PC-1 is dependent upon its phosphodiesterase/pyrophosphatase activity remains controversial. In this study, the extracellular domain of the human PC-1 in its native form or with a T256A or T256S mutation was overexpressed and purified. Enzymatic assays showed that both mutants have less than 10% of the activity of the wild-type protein. In HEK293 cells stably expressing recombinant insulin receptor or insulin-like growth factor 1 (IGF1) receptor, transient expression of wild-type full length PC-1 (PC-1.FL.WT) but not the T256A or T256S mutants inhibits insulin signaling without affecting IGF1 signaling. Western blot and FACS analysis showed that the wild-type and mutant full length PC-1 proteins are expressed at similar levels in the cells, and were localized to the similar levels on the cell surface. Overexpression of PC-1.FL.WT did not affect insulin receptor mRNA level, total protein and cell surface levels. Together, these results suggest that the inhibition of insulin signaling by PC-1 is somewhat specific and is dependent upon the enzymatic activity of the phosphodiesterase/pyrophosphatase.

Override of the osteoclast defect in osteopontin-deficient mice by metastatic tumor growth in the bone.

Osteopontin (OPN) is a major noncollagenous protein of bone that is frequently up-regulated in tumors, where it enhances tumor growth. OPN-deficient mice are resistant to stimulated bone resorption, including that occurring after ovariectomy. Using a new syngeneic model of bone metastasis (r3T), we examined whether OPN-deficient mice are similarly resistant to bone loss resulting from osteolytic tumor growth. Transformed mammary epithelial cells, r3T, which express parathyroid hormone-related protein but not receptor activator of nuclear factor-kappaB ligand, were injected via the intracardiac route into both wild-type and OPN-/- mice. We measured tumor burden in the bone by quantitative polymerase chain reaction assay and evaluated bone loss by X-ray and microCT. Unexpectedly, bone loss was similar in OPN-/- and wild-type mice bearing similar-sized tumors. Osteoclast number was comparable in both genotypes, and the expression of bone sialoprotein was similar in tumor-bearing bones of both genotypes, excluding two potential mechanisms of overriding the defect. Taken together, these results indicate that in the absence of OPN, the bone loss associated with tumor growth at the bone site proceeds rapidly despite the osteoclast defects documented in OPN-/- mice, suggesting that the mechanism of bone loss due to tumor growth differs from that occurring in other pathologies.

A mouse model of breast cancer metastasis to the choroid of the eye.

Transformed mouse mammary epithelial cells, r3T, injected into the arterial circulation form bone metastases with high frequency. Here we report that metastases to the choroid of the eye also occur in these mice with a penetrance of at least 50%. The tumors can occupy as much as half the volume of the eye, and pigmented cells become incorporated into and distributed throughout the tumors. Pigmentation is also observed in the brains and optic nerves of mice with choroidal tumors, suggesting that the tumor cells stimulate migration of pigmented cells along the optic nerve into the brain. To our knowledge, this is the first mouse model of breast cancer choroidal metastasis, and should be useful in the study of this disease.