The klotho gene may be involved in the aging process. Klotho is a coactivator of FGF23, a regulator of phosphate and vitamin D metabolism. It has also been reported to be downregulated in insulin resistance syndromes and paradoxically to directly inhibit IGF-1 and insulin signaling. Our aim was to study klotho's regulation and effects on insulin and IGF-1 signaling to unravel this paradox. We studied klotho tissue distribution and expression by quantitative real-time polymerase chain reaction and Western blotting in obese Zucker rats and high-fat fed Wistar rats, two models of insulin resistance. Klotho was expressed in kidneys but at much lower levels (<1.5%) in liver, muscle, brain, and adipose tissue. There were no significant differences between insulin resistant and control animals. We next produced human recombinant soluble klotho protein (KLEC) and studied its effects on insulin and IGF-1 signaling in cultured cells. In HEK293 cells, FGF23 signaling (judged by FRS2-α and ERK1/2 phosphorylation) was activated by conditioned media from KLECproducing cells (CM-KLEC); however, IGF-1 signaling was unaffected. CM-KLEC did not inhibit IGF-1 and insulin signaling in L6 and Hep G2 cells, as judged by Akt and ERK1/2 phosphorylation. We conclude that decreased klotho expression is not a general feature of rodent models of insulin resistance. Further, the soluble klotho protein does not inhibit IGF-1 and/or insulin signaling in HEK293, L6, and HepG2 cells, arguing against a direct role of klotho in insulin signaling. However, the hypothesis that klotho indirectly regulates insulin sensitivity via FGF23 activation remains to be investigated.

Summary.  Background: Vascular endothelial growth factor (VEGF) and histamine induce von Willebrand factor (VWF) release from vascular endothelial cells. Protein kinase C (PKC) is involved in the control of exocytosis in many secretory cell types. Objectives: We investigated the role of PKC and the interactions between PKC and Ca2+ signaling in both VEGF‐induced and histamine‐induced VWF secretion from human umbilical vein endothelial cells (HUVECs). Results: Several PKC inhibitors (staurosporine, Ro31‐8220, myristoylated PKC peptide inhibitor and Go6983) block VEGF‐induced but not histamine‐induced VWF secretion. PKC‐α and novel PKCs (PKC‐δ, PKC‐ε, and PKC‐η), but not PKC‐β, are expressed in HUVECs. Both VEGF and histamine activate PKC‐δ. However, gene inactivation experiments using small interfering RNA indicate that PKC‐δ (but not PKC‐α) is involved in the regulation of VEGF‐induced but not histamine‐induced secretion. Both VEGF and histamine induce a rise in cytosolic free Ca2+ ([Ca2+]c), but the response to VEGF is weaker and even absent in a significant subset of cells. Furthermore, VEGF‐induced secretion is largely preserved when the rise in [Ca2+]c is prevented by BAPTA‐AM. Conclusions: Our study identifies striking agonist specificities in signal–secretion coupling. Histamine‐induced secretion is dependent on [Ca2+]c but not PKC, whereas VEGF‐induced secretion is largely dependent on PKC‐δ and significantly less on [Ca2+]c. Our data firmly establish the key role of PKC‐δ in VEGF‐induced VWF release, but suggest that a third, VEGF‐specific, signaling intermediate is required as a PKC‐δ coactivator.

The elements of the author's name were wrongly coded in the metadata of this article, with the result that the name was listed incorrectly in all indexing services. His given name is Ulrich M.; his surname, Vischer.The online version of the original article can be found under http://dx.