Hormone-sensitive lipase deficiency disturbs the fatty acid composition of mouse testis.

Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from intracellular stores. In mice, HSL deficiency results in male sterility caused by a major defect in spermatogenesis. The testes contain high concentrations of PUFA and specific PUFA are essential for spermatogenesis. We investigated the fatty acid composition and the mRNA levels of key enzymes involved in fatty acid metabolism in testis of HSL-knockout mice. HSL deficiency altered fatty acid composition in the testis but not in plasma. The most important changes were decreases in the essential n-6 PUFA LNA and the n-3 PUFA ALA, and an increase in the corresponding synthesis intermediates C22:4n-6 and C22:5n-3 without changes in DPAn-6 or DHA acids. Mead acid, which has been associated with an essential fatty acid deficit leading to male infertility, was increased in the testis from HSL-knockout mice. Moreover, the expression of SCD-1, FADS1, and FADS2 was increased while expression of ELOVL2, an essential enzyme for the formation of very-long PUFA in testis, was decreased. Given the indispensability of these fatty acids for spermatogenesis, the changes in fatty acid metabolism observed in testes from HSL-knockout male mice may underlie the infertility of these animals.

Sam68 interacts with IRS1.

Sam68 (Src associated in mitosis) is a RNA binding protein that links cellular signaling to RNA processing. In previous studies we found that insulin promotes Sam68 relocalization in the cytoplasm allowing Sam68 to associate with p85PI3K, Grb2, GAP and probably the insulin receptor (IR), modulating insulin action positively. In the present work, we wanted to define the role of Sam68 in the first stages of IR signaling. Both BRET and co-immunoprecipitation assays have been used for the study of Sam68 binding to IR, IRS1 and p85-PI3K. BRET saturation experiments indicated, for the first time, that Sam68 associates with IRS1 in basal condition. To map the region of Sam68 implicated in the interaction with IRS1, different Sam68 mutants deleted in the proline-rich domains were used. The deletion of P0, P1 and P2 proline rich domains in N-terminus as well as P4 and P5 in C-terminus of Sam68 increased BRET(50), thus indicating that the affinity of Sam68 for IRS1 is lower when these domains are missing. Moreover, in IR-transfected HEK-293 cells, BRET saturation experiment indicated that insulin increases the affinity between Sam68-Rluc and IRS1-YFP. In conclusion, our data indicate that Sam68 interacts with IRS-1 in basal conditions, and insulin increases the affinity between these two partners.

Haloperidol disrupts lipid rafts and impairs insulin signaling in SH-SY5Y cells.

Haloperidol exerts its therapeutic effects basically by acting on dopamine receptors. We previously reported that haloperidol inhibits cholesterol biosynthesis in cultured cells. In the present work we investigated its effects on lipid-raft composition and functionality. In both neuroblastoma SH-SY5Y and promyelocytic HL-60 human cell lines, haloperidol inhibited cholesterol biosynthesis resulting in a decrease of the cell cholesterol content and the accumulation of different sterol intermediates (7-dehydrocholesterol, zymostenol and cholesta-8,14-dien-3beta-ol) depending on the dose of the drug. As a consequence, the cholesterol content in lipid rafts was greatly reduced, and several pre-cholesterol sterols, particularly cholesta-8,14-dien-3beta-ol, were incorporated into the cell membrane. This was accompanied by the disruption of lipid rafts, with redistribution of flotillin-1 and Fyn and the impairment of insulin-Akt signaling. Supplementing the medium with free cholesterol abrogated the effects of haloperidol on lipid-raft composition and functionality. LDL (low-density lipoprotein), a physiological vehicle of cholesterol in plasma, was much less effective in preventing the effects of haloperidol, which is attributed to the drug's inhibition of intracellular vesicular trafficking. These effects on cellular cholesterol homeostasis that ultimately result in the alteration of lipid-raft-dependent insulin signaling action may underlie some of the metabolic effects of this widely used antipsychotic.