Dietary effect of pomegranate seed oil rich in 9cis, 11trans, 13cis conjugated linolenic acid on lipid metabolism in obese, hyperlipidemic OLETF Rats

Conjugated fatty acid, the general term of positional and geometric isomers of polyunsaturated fatty acids with conjugated double bonds, has attracted considerable attention because of its potentially beneficial biological effects. In the present study, dietary effect of pomegranate seed oil rich in punicic acid (9cis, 11trans, 13cis-conjugated linolenic acid; 9c, 11t, 13c-CLNA) on lipid metabolism was investigated in obese, hyperlipidemic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. After 2 weeks feeding period, OLETF rats revealed obesity and hyperlipidemia compared with their progenitor LETO rats. Feeding of the diet supplemented with 9% safflower oil and 1% pomegranate seed oil (9c, 11t, 13c-CLNA diet) did not affect abdominal white adipose tissue weights and serum lipid levels compared with the diet supplemented with 10% safflower oil (control diet) in OLETF rats. However, the accumulated hepatic triacylglycerol was markedly decreased by 9c, 11t, 13c-CLNA diet in OLETF rats. Activities of hepatic enzymes related to fatty acid synthesis and fatty acid β-oxidation were not altered by 9c, 11t, 13c-CLNA diet. Levels of monounsaturated fatty acid (MUFA), major storage form of fatty acid, in serum triacylglycerol were markedly higher in obese, hyperlipidemic OLETF rats than in lean LETO rats. In addition, 9c, 11t, 13c-CLNA diet significantly decreased MUFA levels in OLETF rats. This is the first study showing that 9c, 11t, 13c-CLNA suppresses delta-9 desaturation in vivo, and we suggest that the alleviation of hepatic triacylglycerol accumulation by 9c, 11t, 13c-CLNA diet was, at least in part, attributable to the suppression of delta-9 desaturation in OLETF rats

Karyopherin alpha2 is essential for rRNA transcription and protein synthesis in proliferative keratinocytes.

Karyopherin proteins mediate nucleocytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest KPNA2 expression is induced in tumor cells and is strongly associated with prognosis, although the precise roles and mechanisms of KPNA2 overexpression in proliferative disorders have not been defined. We found that KPNA2 expression is induced in various proliferative disorders of the skin such as psoriasis, Bowen's disease, actinic keratosis, squamous cell carcinoma, Paget's disease, Merkel cell carcinoma, and mycosis fungoides. siRNA-mediated KPNA suppression revealed that KPNA2 is essential for significant suppression of HaCaT proliferation under starvation conditions. Ribosomal RNA transcription and protein synthesis were suppressed by starvation combined with knockdown of KPNA (including KPNA2) expression. KPNA2 localized to the nucleolus and interacted with proteins associated with mRNA processing, ribonucleoprotein complex biogenesis, chromatin modification, and transcription, as demonstrated by tandem affinity purification and mass spectrometry. KPNA2 may be an important promoter of ribosomal RNA and protein synthesis in tumor cells

Lists of proteins analyzed by pathway analysis.

<p>Lists of proteins analyzed by pathway analysis.</p

Suppression of protein synthesis by combined KPNA knockdown.

<p>Under starvation conditions (0.1% fetal bovine serum), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 significantly suppressed protein synthesis after 48 h (*p<0.05) and 72 h (***p<0.01), as demonstrated by metabolic labeling with ³⁵S-methionine.</p

Detection and analysis of proteins that interact with KPNA2 and localization of KPNA2 in the nucleolus.

<p>Proteins that interact with KPNA2 in the cytoplasm and nucleus were purified using the TAP method and detected by silver staining. Proteins marked with arrows were analyzed by LC/MS/MS. HaCaT cells expressing GFP-TAP were used to detect nonspecific interactions. <b>a)</b> The results of LC/MS/MS were analyzed by pathway analysis using reactome (<a href="http://www.reactome.org" target="_blank">http://www.reactome.org</a>). The categories of “mRNA processing”, “ribonucleoprotein complex biogenesis”, “chromatin modification,” and “transcription” were the most significantly represented pathways. <b>b)</b> Immunohistochemistry revealed KPNA2 co-localization with UBF, a nucleolar marker.</p

Suppression of ribosomal RNA synthesis by combined KPNA knockdown.

<p>Under starvation conditions (0.1% fetal bovine serum), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 significantly suppressed ribosomal RNA synthesis analyzed by reverse transcription-quantitative polymerase chain reaction (***p<0.01). The amount of pre-ribosomal RNA was reduced by about 37% after 72 h.</p

Suppression of cell growth by combined KPNA knockdown.

<p>Under starvation conditions (0.1% FBS), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 suppressed cell growth after 120 h (*p<0.05). Only KPNA2 siRNA subtraction produced no change in proliferation.</p