Lipid synthesis and secretion by primary cultures of rat mammary epithelial cells

Lipid synthesis and secretion was measured in primary rat mammary epithelial cells cultured on basement matrix in medium supplemented with lactogenic hormones. The cells grew and differentiated to form alveolar‐like structures reminiscent of lactating mammary gland. They synthesized abundant triacylglycerol, containing fatty acids characteristic of rat milk (C10:O‐C14:0), using 14C‐glucose, 14C‐oleic acid or 14C‐glycerol as precursors. Basal levels of triacylglycerol secretion were measured using 14C‐oleic acid labeling; 1.3±0.3% of the labeled cellular triacylglycerol was secreted into the medium in 24 hours. Secreted lipid droplets were surrounded by a bilayer membrane with an electron‐dense inner coat characteristic of fat globules secreted by the mammary gland. The rate of triglycerol secretion was increased to 998±98% of control (P<0.01) by the addition of phorbol 12‐myristate 13‐acetate (PMA) in combination with staurosporine, a protein kinase inhibitcn. Several other protein kinase inhibitors, when combined with PMA, also markedly stimulated secretion. Effective protein kinase inhibitors included sphingosine (has diverse cellular effects including the inhibition of protein kinase C; 13‐fold increase in secretion), and KT5823 (a cGMP dependent protein kinase inhibitor; 5‐fold increase). KT5720 (a cAMP‐dependent protein kinase inhibitor) did not alter secretion. Kinase inhibitors were effective only in the presence of a phorbol ester. 4α‐phorbol‐12,13‐didecanoate, a phorbol ester which does not activate protein kinase C (PKC), could substitute for PMA. Lipid release was not mediated by disruption of cell‐cell tight junctions, as EGTA did not release lipid. Based on these observations we suggest that two signals are needed to enable or stimulate lipid secretion in cultured rat mammary epithelial cells: (1) inhibition of a protein kinase and (2) a PKC‐independent effect of phorbol ester. We have, for the first time, characterized a cell culture model suitable for studying lipid synthesis and secretion by mammary epithelial cells

Temperature-Responsive Nanospheres with Bicontinuous Internal Structures from a Semicrystalline Amphiphilic Block Copolymer

Internally structured self-assembled nanospheres, cubosomes, are formed from a semicrystalline block copolymer, poly(ethylene oxide)-block-poly(octadecyl methacrylate) (PEO39-b-PODMA17), in aqueous dispersion. The PODMA block provides them with a temperature-responsive structure and morphology. Using cryo-electron tomography, we show that at room temperature these internally bicontinuous aggregates undergo an unprecedented order?disorder transition of the microphase-separated domains that is accompanied by a change in the overall aggregate morphology. This allows switching between spheres with ordered bicontinuous internal structures at temperatures below the transition temperature and more planar oblate spheroids with a disordered microphase-separated state above the transition temperature. The bicontinuous structures offer a number of possibilities for application as templates, e.g., for biomimetic mineralization or polymerization. Furthermore, the unique nature of the thermal transition observed for this system offers up considerable possibilities for their application as temperature-controlled release vessels