In the present study, we investigated a putative function of OSBP-related proteins in the regulation of serum lipids and lipoproteins in humans. Genotyping of OSBPL
gene SNPs in a dyslipidemic family material was initially used to evaluate the contribution of OSBPL
genes to extreme lipid levels predisposing to cardiovascular disease, high triglycerides, and low HDL. Although recent genome-wide association studies [21
] have initially identified common variants affecting lipid concentrations at the population level, data are also emerging for the impact of multiple rare variants influencing the extreme lipid levels [24
]. Here, we wanted to study such potential loci by testing the involvement of select OSBPL
genes in carefully phenotyped Finnish families ascertained for dyslipidemias, low HDL, or FCHL. The FCHL study set revealed suggestive linkage of several SNPs in OSBPL10
to the high TG trait, and analysis of the combined material strengthened the evidence. Importantly, association analysis using an independent study sample consisting of subjects with metabolic syndrome and matched healthy controls provided substantial proof for association of OSBPL10
SNPs with the extreme end (>95th percentile) high-TG trait.
The genetic analyses suggest that variation within the chromosome 3p23 region containing OSBPL10 may contribute to the high TG levels in Finnish dyslipidemic subjects. The present data do not allow us to firmly conclude that the underlying gene is OSBPL10, but they prompted us to investigate in more detail the function of the encoded protein, ORP10, in cultured cell models. The metabolic studies in cultured cells subjected to ORP10 silencing identify the protein as a novel regulator of hepatocellular lipid metabolism and apoB100 secretion, consistent with the notion that OSBPL10 may be the gene underlying the genetic findings. The likelihood of this is further increased by the lack of other genes plausibly connected with lipid metabolism within the chromosome region (10 Mb interval).
Hepatoma cells subjected to ORP10 silencing displayed a significant increase in the incorporation of [3
H]acetate into cholesterol and of [3
H]acetate and [3
H]oleic acid into TG, suggesting that the intrinsic function of ORP10 involves suppression of hepatocellular lipid biosyntheses. Consistent with this observation, the cells showed increased HMG-CoA reductase activity, demonstrating an effect already at an early stage of the pathway, on conversion of HMG-CoA to mevalonic acid. An independent, systematic esiRNA silencing screen of all ORP family members carried out in HeLa cells supported a role of ORP10 in the regulation of cholesterol biosynthesis (data not shown). Labeling with [3
H]oleic acid suggested that the impact of ORP10 on TG biosynthesis occurs at the levels of fatty acid activation or acyl transfer to glycerol. Importantly, we also found that ORP10 silencing enhances the accumulation of apoB100 in the hepatoma cell growth medium. One can envision that ORP10 could affect the synthesis of TG and cholesterol as well as the assembly of TG-rich lipoproteins in the intestine and the liver, both tissues in which OSBPL10
mRNA is expressed abundantly [4
ORP10 was found to distribute between a cytosolic localization and microtubules, a major apparatus determining the spatial distribution of organelles and forming tracks for intracellular transport events [25
]. FRAP analysis of live Huh7 cells with EGFP-ORP10 on microtubules revealed a recovery with two kinetic components. We find it likely that the fast component (t1/2
4.5 s) reflects the diffusion of EGFP-ORP10 in the cytosolic compartment, while the slow component (t1/2
64.3 s) could represent the actual microtubule association of the protein. The latter half-time is in the same range as the life-times determined for transport carriers moving along microtubules from the endoplasmic reticulum (ER) to the Golgi complex [26
] and from the trans
-Golgi network to the plasma membrane [27
]. The data are thus consistent with the notion that ORP10 could be involved in microtubule-dependent membrane trafficking or organelle motility.
Microtubule-dependent membrane trafficking is intimately connected with the control of lipid metabolism. Importantly, the membrane compartments that play key roles in the cellular metabolism of cholesterol and triglycerides, the ER and cytoplasmic lipid droplets, are connected to microtubules, which determine the spatial organization of the ER [29
] and control the distribution and motility of lipid droplets [30
]. Pertinent to serum lipoproteins, also the Golgi complex playing a central role in lipoprotein secretion is organized by microtubules [32
]. One can therefore envision that a protein associated with microtubules, such as ORP10, is in the position to modulate cellular lipid metabolism and lipoprotein secretion.