Hepatic STAMP2 alleviates high fat diet-induced hepatic steatosis and insulin resistance.

BACKGROUND & AIMS: Most studies on the role of STAMP2 in metabolism have used adipose tissue. Little knowledge exists concerning the role of STAMP2 in the liver, which is a metabolically central target. We hypothesized that STAMP2 is involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis. METHODS: We examined our hypothesis using human NAFLD patient pathology samples and a high-fat diet (HFD)-induced NAFLD mouse model. The molecular mechanism underlying hepatic STAMP2-mediated lipid imbalance was explored using an oleic acid (OA)-induced NAFLD in vitro model. RESULTS: Noticeably, the expression level of STAMP2 protein was reduced in the livers obtained from NAFLD patients and HFD-induced NAFLD mice. In vivo knockdown of hepatic STAMP2 by siRNA accelerated hepatic steatosis and insulin resistance in mice fed a HFD. Conversely, the delivery of adenoviral STAMP2 (Ad-STAMP2) improved hepatic steatosis in HFD-induced NAFLD mice. The expression of lipogenic or adipogenic factors was increased in both in vitro and in vivo NAFLD models but was reversed by Ad-STAMP2. Adenoviral overexpression of STAMP2 improved insulin resistance in the HFD-induced NAFLD mice. In vivo and in vitro assays demonstrated that STAMP2 modulates insulin sensitivity and glucose metabolism and that STAMP2 counteracts OA-induced insulin resistance by modulating insulin receptor substrate-1 stability. CONCLUSIONS: The present study revealed that hepatic STAMP2 plays a pivotal role in preventing HFD-induced NAFLD and that STAMP2 overexpression improves hepatic steatosis and insulin resistance in NAFLD. Our findings indicate that STAMP2 may represent a suitable target for interventions targeting NAFLD.

Early mitochondrial hyperpolarization and intracellular alkalinization in lactacystin-induced apoptosis of retinal pigment epithelial cells.

We investigated the induction and underlying mechanism of apoptosis in retinal pigment epithelial cells by the inhibition of proteasome activity using lactacystin. Rat retinal pigment epithelial cell line retinal pigment epithelial (RPE)-J was used in this study. Apoptosis was evaluated by light and electron microscopies, DNA electrophoresis, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The apoptosis-related proteins were localized in the cells by immunofluorescent microscopy, and the changes of their protein contents and the enzyme activation were monitored by Western blot. Mitochondrial membrane potential was quantified by measuring J aggregate (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol carbocyanine iodide) fluorescence. To measure changes in intracellular pH, cells were loaded with 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein and assayed by flow cytometry. To elucidate the type of transport system involving intracellular pH regulation, several transporter inhibitors were used, and their effect on pH and membrane potential was assayed as described above. Lactacystin treatment significantly induced apoptosis in RPE-J cells. During the RPE cell apoptosis, 1) cytochrome c and Smac/DIABLO were released into cytosol from mitochondria, 2) translocation of apoptosis-inducing factor to the nucleus was evident, 3) Bax protein seemed to translocate to mitochondria, 4) procaspase-3 and poly(ADP-ribose) polymerase were cleaved, and 5) nuclear condensation and DNA fragmentation were clearly observed. Noticeably, a transient increase of mitochondrial membrane potential was coincidentally detected with the intracellular alkalinization after lactacystin administration. Furthermore, the lactacystin-induced early alkalinization was inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate, an inhibitor of Cl(-)/HCO(3)(-) anion exchanger, which also prevented early mitochondrial hyperpolarization and apoptosis. Lactacystin-induced apoptosis in RPE-J cells is closely associated with an early mitochondrial hyperpolarization induced by intracellular alkalinization.