RESUMO
Establishment and maintenance of pregnancy depends on progesterone synthesized by luteal tissue in the ovary. Our objective was to identify the characteristics of lipid droplets (LDs) in ovarian steroidogenic cells. We hypothesized that LDs are a major feature of steroidogenic luteal cells and store cholesteryl esters. Whole bovine tissues, isolated ovarian steroidogenic cells (granulosa, theca, small luteal, and large luteal), and isolated luteal LDs were assessed for LD content, LD-associated proteins and lipid analyses. Bovine luteal tissue contained abundant lipid droplets, LD-associated perilipins 2/3/5, hormone-sensitive lipase, and 1-acylglycerol-3-phosphate O-acyltransferase ABHD5. Luteal tissue was enriched in triglycerides (TGs) compared to other tissues, except for adipose tissue. Luteal cells were distinguishable from follicular cells by the presence of LDs, LD-associated proteins, and increased TGs. Furthermore, LDs from large luteal cells were numerous and small; whereas, LDs from small luteal cells were large and less numerous. Isolated LDs contained nearly all of the TGs and cholesteryl esters present in luteal tissue. Isolated luteal LDs were composed primarily of TG, with lesser amounts of cholesteryl esters, diglyceride and other phospholipids. Bovine luteal LDs are distinct from LDs in other bovine tissues, including follicular steroidogenic cells.
Assuntos
Corpo Lúteo/metabolismo , Gotículas Lipídicas/química , Lipídeos/química , Ovário/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/química , Animais , Bovinos , Ésteres do Colesterol/metabolismo , Feminino , Células da Granulosa/metabolismo , Lipidômica , Células Lúteas/metabolismo , Microscopia Confocal , Ovulação , Perilipina-1/química , Progesterona/metabolismo , Espectrometria de Massas em Tandem , Células Tecais/metabolismoRESUMO
Perilipins (PLINs) are proteins that associate with lipid droplets (LDs) and play roles in the control of triglycerides (TG) metabolism. Two types of PLINs - 1 and 2- occur in insects. Following previous work on MsPLIN1A (a 42â¯kDa protein formerly called MsLsd1), here we report a new PLIN1 isoform, MsPLIN1B. MsPLIN1B cDNA was cloned and the 1835bp cDNA contains an ORF encoding a 47.9â¯kDa protein whose expression was confirmed by mass spectrometry. Alternative transcripts A and B, which differ in the alternative use of exon 1, were the most abundant PLIN1 transcripts in the fat body. These transcripts encode nearly identical proteins except that the B isoform contains 59 additional residues in its amino terminus. No conserved domain was identified in the extra region of MsPLIN1B. The novel PLIN1 isoform is found in lepidopteran species. In Manduca, PLIN1B was expressed only in the 5th instar larva and its levels correlated with fat storage. Furthermore, PLIN1B levels increased with the fat content of the diet in insects of the same age confirming a direct relationship between PLIN1B and TG storage irrespective of development. The nutritional status impacted PLIN1B levels, which decreased in starvation and increased with subsequent re-feeding. Altogether data support a link between PLIN1B and TG storage in caterpillars prior to pupation. The combined findings suggest distinct roles for PLIN1A, PLIN1B and PLIN2. MsPLIN1A abundance correlates with mobilization of TG stores, MsPLIN2 with the synthesis of new LDs and MsPLIN1B abundance correlates with high levels of TG storage and large LD sizes at the end of the last feeding period.
Assuntos
Corpo Adiposo/metabolismo , Proteínas de Insetos/genética , Manduca/genética , Perilipina-1/genética , Sequência de Aminoácidos , Animais , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Metabolismo dos Lipídeos/genética , Manduca/crescimento & desenvolvimento , Manduca/metabolismo , Perilipina-1/química , Perilipina-1/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de SequênciaRESUMO
Lipid droplets are "small" organelles that play an important role in de novo synthesis of new membrane, and steroid hormones, as well as in energy storage. The way proteins interact specifically with the oil-(phospho-)lipid monolayer interface of lipid droplets is a relatively unexplored but crucial question. Here, we use our home built liquid droplet tensiometer to mimic intracellular lipid droplets and study protein-lipid interactions at this interface. As model neutral lipid binding protein, we use apoLp-III, an amphipathic α-helix bundle protein. This domain is also found in proteins from the perilipin family and in apoE. Protein binding to the monolayer is studied by the decrease in the oil/water surface tension. Previous work used POPC (one of the major lipids found on lipid droplets) to form the phospholipid monolayer on the triolein surface. Here we expand this work by incorporating other lipids with different physico-chemical properties to study the effect of charge and lipid head-group size. This study sheds light on the affinity of this important protein domain to interact with lipids.