An Overview of Homocysteine Metabolism - Homocysteine in Protein Structure/Function and Human Disease - page 14

developmental abnormalities. In one study Hcy-thiolactone was reported to be non-
teratogenic in mouse embryos, but the maximum dose used in that study [160] was
lower than those used in other studies, so that a teratogenic dose has not been
reached.
Rigorous studies in mice using the intraperitoneal injection model show that
plasma Hcy-thiolactone is cleared with a half-life of 5.0 min [140, 141]. Studies
with Blmh / and Pon1 / mouse models that have diminished ability to hydrolyze
Hcy-thiolactone led to a conclusion that bleomycin hydrolase (Blmh) and
paraoxonase 1 (Pon1) protect the animals against neurotoxicity induced by intra-
peritoneal injections of L -Hcy-thiolactone [140, 141]. Similar to other harmful
products of normal metabolism, endogenous Hcy-thiolactone is also eliminated
by urinary excretion, as demonstrated both in humans and mice [93, 95].
Treatments with Hcy-thiolactone inhibit proliferation and can be toxic to
cultured human and animal cells [143]. For instance, short-term pretreatment
(10 min) of cultured rat hepatoma cells with Hcy-thiolactone leads to the inhibition
of insulin signaling by preventing tyrosine phosphorylation of the -subunit of the

insulin receptor and its substrates IRS-1 and Sam68 [161]. Furthermore,
pretreatment with Hcy-thiolactone inhibits the interactions of the insulin receptor
β

β

-subunit and its substrates with the regulatory subunit p85 of phosphatidylinositol
3-kinase, which in turn inhibits the kinase and blocks the insulin-stimulated glyco-
gen synthesis [161]. Hcy-thiolactone also prevents phosphorylation of GSK-3 and
p70S6K catalyzed by mitogen-activated protein kinase, as well as insulin-mediated
growth and proliferation [162]. The addition of GSH 5 min before the pretreatment
with Hcy-thiolactone restores insulin signaling, suggesting that Hcy-thiolactone-
induced oxidative stress might be involved [161, 162]. Much longer (24 h)
treatments with higher Hcy concentrations (0.1-1 mM) induce insulin resistance
by reducing tyrosine phosphorylation of insulin receptor and IRS-1 in rat
adipocytes [163]. Treatments with 0.3-1 mM Hcy (lower concentration is ineffec-
tive) also induce resistin, a peptide hormone linked to insulin resistance, after
8-24 h (no induction after 4 h) [163]. Taken together, these findings suggest that
Hcy-thiolactone is more effective than Hcy in inducing insulin resistance in
cultured cells and thus is likely to contribute of insulin resistance associated with
hyperhomocysteinemia in humans and experimental animals.
Hcy-thiolactone is also known to induce endoplasmic reticulum (ER) stress and
unfolded protein response (UPR) in retinal epithelial cells [60], as well as apoptotic
death in cultured human vascular endothelial cells [61, 164], promyeloid cells
[165], and placental trophoblasts [166]. For example, human umbilical vein endo-
thelial cells (HUVECs) treated with 50-200 M D , L -Hcy-thiolactone for 24 h show

15-40 % apoptotic cell death quantified after labeling with the fluorescent probe
DiOC6(3) and flow cytometry analysis (Table 2.2 ) [154]. The magnitude of apo-
ptosis in HUVECs treated with 200

μ

M D , L -Hcy-thiolactone corresponds to about
2/3 of apoptosis observed in the cells treated with 5

μ

g/mL staurosporine or serum-
free medium as positive controls (Table 2.2 ). In contrast to apoptosis induced by
staurosporine, apoptotic cell death induced by Hcy-thiolactone in HUVECs is not
accompanied by the induction of caspase activity and is not prevented by caspase

μ