Temporal and spatial variations of lipid droplets during adipocyte division and differentiation

By capturing time-lapse images of primary stromal-vascular cells (SVCs) derived from rat mesenteric adipose tissue, we revealed temporal and spatial variations of lipid droplets (LDs) in individual SVCs during adipocyte differentiation. Numerous small LDs (a few micrometers in diameter) appeared in the perinuclear region at an early stage of differentiation; subsequently, several LDs grew to more than 10 μm in diameter and occupied the cytoplasm. We have developed a method for the fluorescence staining of LDs in living adipocytes. Time-lapse observation of the stained cells at higher magnification showed that nascent LDs (several 100 nm in diameter) grew into small LDs while moving from lamellipodia to the perinuclear region. We also found that adipocytes are capable of division and that they evenly distribute the LDs between two daughter cells. Immunofluorescence observations of LD-associated proteins revealed that such cell divisions of SVCs occurred even after LDs were coated with perilipin, suggesting that the “final” cell division during adipocyte differentiation occurs considerably later than that characterized in 3T3-L1 cells. Our time-lapse observations have provided a detailed account of the morphological changes that SVCs undergo during adipocyte division and differentiation. By capturing time-lapse images of primary stromal-vascular cells (SVCs) derived from rat mesenteric adipose tissue, we revealed temporal and spatial variations of lipid droplets (LDs) in individual SVCs during adipocyte differentiation. Numerous small LDs (a few micrometers in diameter) appeared in the perinuclear region at an early stage of differentiation; subsequently, several LDs grew to more than 10 μm in diameter and occupied the cytoplasm. We have developed a method for the fluorescence staining of LDs in living adipocytes. Time-lapse observation of the stained cells at higher magnification showed that nascent LDs (several 100 nm in diameter) grew into small LDs while moving from lamellipodia to the perinuclear region. We also found that adipocytes are capable of division and that they evenly distribute the LDs between two daughter cells. Immunofluorescence observations of LD-associated proteins revealed that such cell divisions of SVCs occurred even after LDs were coated with perilipin, suggesting that the “final” cell division during adipocyte differentiation occurs considerably later than that characterized in 3T3-L1 cells. Our time-lapse observations have provided a detailed account of the morphological changes that SVCs undergo during adipocyte division and differentiation. Adipose tissue, consisting mainly of adipocytes, functions as an organ for energy storage. However, hypertrophy and hyperplasia of adipocytes in the adipose tissue are associated with obesity, a major risk factor for lifestyle-related diseases. Adipocyte proliferation and differentiation have therefore been investigated in various disciplines, such as cell biology, physiology, and pathology. For the past 30 years, 3T3-L1 cells (1Vissers M.N. Zock P.L. Meijer G.W. Katan M.B. Effect of plant sterols from rice bran oil and triterpene alcohols from sheanut oil on serum lipoprotein concentrations in humans..Am. J. Clin. Nutr. 2000; 72: 1510-1515Crossref PubMed Scopus (91) Google Scholar, 2Miettinen T.A. Tilvis R.S. Kesaniemi Y.A. Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population..Am. J. Epidemiol. 1990; 131: 20-31Crossref PubMed Scopus (556) Google Scholar) and primary stromal-vascular cells (SVCs), which are derived from inguinal, abdominal, perirenal, retroperitoneal, and epididymal adipose tissues (3Tikkanen M.J. Plant sterols and stanols. In Handbook of Experimental Pharmocology. Springer-Verlag, Berlin2005: 215-230Google Scholar, 4Bosner M.S. Lange L.G. Stenson W.F. Ostlund Jr., R.E. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry..J. Lipid Res. 1999; 40: 302-308Abstract Full Text Full Text PDF PubMed Google Scholar, 5Sudhop T. Lutjohann D. Kodal A. Igel M. Tribble D.L. Shah S. Perevozskaya I. von Bergmann K. Inhibition of intestinal cholesterol absorption by ezetimibe in humans..Circulation. 2002; 106: 1943-1948Crossref PubMed Scopus (0) Google Scholar, 6Heinemann T. Axtmann G. von Bergmann K. Comparison of intestinal absorption of cholesterol with different plant sterols in man..Eur. J. Clin. Invest. 1993; 23: 827-831Crossref PubMed Scopus (348) Google Scholar, 7Lutjohann D. Bjorkhem I. Beil U.F. von Bergmann K. Sterol absorption and sterol balance in phytosterolemia evaluated by deuterium-labeled sterols: effect of sitostanol treatment..J. Lipid Res. 1995; 36: 1763-1773Abstract Full Text PDF PubMed Google Scholar, 8Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters..Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1328) Google Scholar, 9Lee M.H. Lu K. Hazard S. Yu H. Shulenin S. Hidaka H. Kojima H. Allikmets R. Sakuma N. Pegoraro R. et al.Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption..Nat. Genet. 2001; 27: 79-83Crossref PubMed Scopus (580) Google Scholar), have been reported to be capable of differentiation into adipocytes. Hence, these cells have been used as in vitro models to clarify cellular and molecular events involved in adipocyte differentiation. According to previous reports, preadipocytes can proliferate until they undergo growth arrest at confluence; subsequently, the growth-arrested cells undergo additional cell division, known as clonal expansion, before they start accumulating lipid droplets (LDs) in their cytoplasm (as reviewed in Ref. 10Patel S.B. Salen G. Hidaka H. Kwiterovich P.O. Stalenhoef A.F. Miettinen T.A. Grundy S.M. Lee M.H. Rubenstein J.S. Polymeropoulos M.H. et al.Mapping a gene involved in regulating dietary cholesterol absorption. The sitosterolemia locus is found at chromosome 2p21..J. Clin. Invest. 1998; 102: 1041-1044Crossref PubMed Scopus (157) Google Scholar). Furthermore, other reports have shown that fully mature adipocytes having large, single LD, referred to as unilocular adipocytes, can also proliferate under specific culture conditions, such as “ceiling culture” (11Bjorkhem I. Boberg K.M. Leitersdorf E. Inborn errors in bile acid biosynthesis and storage of sterols other than cholesterol.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. In The Metabolic Bases of Inherited Disease. McGraw-Hill, New York2001: 2961-2988Google Scholar) or “three-dimensional collagen gel culture” (12Katan M.B. Grundy S.M. Jones P. Law M. Miettinen T. Paoletti R. Efficacy and safety of plant stanols and sterols in the management of blood cholesterol levels..Mayo Clin. Proc. 2003; 78: 965-978Abstract Full Text Full Text PDF PubMed Google Scholar). However, it has never been clarified whether adipocytes divide during adipocyte differentiation after they start accumulating LDs, although they have been considered to lack proliferative activity at this stage. In optical microscopy, LDs appear as well-defined spheres. Accumulation of LDs is widely believed to be the most characteristic function of adipocytes. Observations of LDs using electron microscopy reveal that each is by a Meijer G.W. Plant and of and concentrations in and J. Clin. Nutr. 1998; PubMed Scopus Google Scholar), and this that LDs are from the in cells H. Comparison of the of plant sterol and plant in serum cholesterol concentrations in on a J. Clin. Nutr. 2000; PubMed Scopus Google Scholar). has also been revealed that LDs J. T. P. E. J. of serum plant sterols and cholesterol in and of and in and their 40: Full Text PDF PubMed Scopus Google Scholar) and that the of these the of LDs T. von Bergmann K. Serum plant sterols as a risk factor for 2002; Full Text PDF PubMed Scopus Google Scholar). In molecular and various such as adipose also known as perilipin, and have been found in on the the of LDs G. P. J. H. are associated with an of events in of a of the Full Text Full Text PDF PubMed Scopus Google Scholar, H. Miettinen T.A. of serum and sterols with in 2000; PubMed Scopus Google Scholar, Yu L. G. Grundy S. Hobbs H.H. Plant sterol are associated with in and PubMed Scopus Google Scholar, N. S. R. S. A. N. and of the of J. 1999; Google as reviewed in of the of lipoprotein cholesterol in of the PubMed Scopus Google Scholar, G. von Bergmann K. Lutjohann D. Kwiterovich P. J. S.B. T. P. B. plant sterols in with PubMed Scopus Google Scholar). has been reported that the of LD-associated proteins from to in 3T3-L1 cells occurs after the of adipocyte differentiation G. P. J. H. are associated with an of events in of a of the Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, these proteins have been reported to in the and of LDs R.S. Miettinen T.A. Serum plant sterols and their to cholesterol J. Clin. Nutr. PubMed Scopus Google Scholar, M. cholesterol absorption and cholesterol synthesis with Scholar, H. M. J. J. M. R. Miettinen T.A. in the and with cholesterol absorption and Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Plant sterol and and 2000; PubMed Google Scholar). it is also the molecular events the at specific of adipocyte differentiation that have In temporal and spatial variations of the LDs in individual cells the of adipocyte differentiation have never been microscopy and microscopy the of and temporal variations of LDs in cells be the other optical microscopy the observation of cells the of adipocyte differentiation the of cells is Time-lapse observation the of and the involved in the of this of observation can whether adipocytes divide during adipocyte differentiation. In the we time-lapse images of SVCs derived from rat mesenteric adipose tissue the of adipocyte differentiation. The time-lapse observations temporal and spatial variations of LDs in individual cells. Numerous small LDs (a few micrometers in diameter) appeared in the perinuclear region at an early stage of after which several LDs the cytoplasm with several LDs than 10 μm in Furthermore, we developed a method for fluorescence staining of LDs in living adipocytes, and we the temporal and spatial variations of LDs at a higher The revealed that nascent LDs (several 100 nm in diameter) developed in the lamellipodia and grew into small LDs while moving the perinuclear region. Our time-lapse observations also showed that for the of adipocyte differentiation from SVCs and that adipocytes were capable of In such cell division, adipocytes evenly LDs between two daughter cells. Immunofluorescence observations of LD-associated proteins revealed that adipocyte cell division occurred even after the LDs were coated with that the “final” cell division during adipocyte differentiation from primary SVCs occurs considerably later than that characterized in 3T3-L1 cells. We used a primary culture Adipocyte a from the The culture SVCs derived from the mesenteric adipose tissue of and a culture on by et S.M. R. factor and the risk of or to in or the J. Full Text Full Text PDF PubMed Scopus Google Scholar). In their the culture with 100 10 100 and 100 as for the of adipocyte differentiation from SVCs were in the for more than of the cells the adipocyte with an of LDs in their cytoplasm. We used this culture the of adipocyte differentiation in For cell SVCs were in a the from the cell we the The with the culture after at for and the cells were The cell at an of into and a of to each The in which cells were were at and in a during the cell culture an the with culture to the The of culture reported to be an method for time-lapse observations of cells P. H. L. of and in to and in 40: Full Text PDF PubMed Scopus Google Scholar). For the time-lapse observations of SVCs during adipocyte differentiation (several we to the culture to and and a lipid and oil at to the The of lipid reported to the of LDs during adipocyte differentiation S.M. R. factor and the risk of or to in or the J. Full Text Full Text PDF PubMed Scopus Google Scholar), although the lipid for adipocyte differentiation. The with the culture with the lipid and with a and it to the the the of adipocytes have in the of a in to the of In the we that the of the by in a that the cells were with a of Time-lapse observations were using a or a with a and a The of the the the and the in an in which the at on the which time-lapse images were The of images into a using and (a from K. were used for the fluorescence staining of LDs and in living cells. a specific for LDs K.E. von Bergmann K. Lutjohann D. R. Grundy S.M. Hobbs H.H. of sterols and to in and Lipid Res. 2002; Full Text Full Text PDF PubMed Google Scholar), in to a to the method used in a previous G. Jr., Grundy S.M. of in Clin. Invest. PubMed Scopus Google Scholar). in to a were to the culture at concentrations of and The cells were stained in a for the 10 of the cell culture The stained cells were with culture The as that the lipid to the The that we used for time-lapse observations with an additional oil and for fluorescence The by images of and were by to the to the fluorescence images also of images were fluorescence images of and were to a single and the of images on images into a as SVCs were in and were in the culture for after they were in the culture with lipid various after the the cells were with and with in for 10 and they were with The cells were with in for 10 and with and were with for 30 The cells were with primary in for and were with The cells were with in for and were additional by using as the primary and as the with as the primary and as the the cell with and in cells were with for 10 after the with images and images were using the that used for fluorescence observations of the morphological changes that SVCs undergo during adipocyte we used microscopy to time-lapse images of SVCs in The observation after the cells were in the and it for time-lapse images of SVCs are shown in The were into a in the the cells and on the and of the with the cells. a cells the adipocyte as by the of LDs in after the most cells a and and cells LDs on these it can be considered that the cells with LDs are from the SVCs in the 100 cells by cell division and into adipocytes. The LDs that in each adipocyte grew from a few micrometers to more than 10 μm in diameter during this observations are with a previous S.M. R. factor and the risk of or to in or the J. Full Text Full Text PDF PubMed Scopus Google Scholar). adipocytes as as cells showed on the The adipocytes to with each other as to a or a In the of the observation an of adipocytes in 100 of the with adipocytes and other cells. the of LDs in we spatial and temporal variations of the LDs in individual cells the of adipocyte differentiation. The observation using microscopy the of between LDs more than that using temporal and spatial variations of the LDs in individual cells also in the LDs, which were a few micrometers in appeared in the perinuclear region at to after For the the of LDs the of each showed the cell the with to a in the cells from and LDs to and In this the to the cell to the LDs that occupied the of the cell Furthermore, a morphological in cell as the cells from a of to a of by the of LDs μm in diameter) occupied the region of the and were this region at the LDs to with to the in of the small LDs and the were in the region we temporal and spatial variations of the LDs in other the at which small LDs appeared in the perinuclear region to found to be cells in after The of the in individual cells found to be cells in clarify LDs we time-lapse observations of adipocytes at higher magnification In this cells of it also to LDs from other these we fluorescence staining of LDs in living adipocytes. a fluorescence and a Numerous than μm in diameter) were in the fluorescence and these were with the well-defined in the that LDs can be stained in living adipocytes. The LDs were in the cell in or in the perinuclear region in LDs were as cells or and spatial variations of nascent LDs in a adipocyte are shown in in the LDs developed the lamellipodia during the observation The LDs were or the of the LDs in developed into small they the the small LDs We that adipocytes are capable of division even during the of differentiation. time-lapse images of the cell division in the to after 30 an by in LDs to a few micrometers in diameter) in the cell 30 to the adipocyte a The adipocyte into two daughter cells the 30 LDs were also in the daughter cells after the cell the individual cells on the and in different LDs were in daughter cell and LDs were in the we the of LDs in the of cell division, the of the of LDs in the two daughter cells to that in the cell found to be the of the of LDs in daughter cell to that in the cell found to be daughter that LDs in a adipocyte are evenly between two daughter cells or In of the adipocytes that accumulating LDs were to divide into two daughter cells. it that the at which these adipocytes cell division from to after cells were also to undergo cell The adipocyte shown in also in a before the images were adipocytes were capable of division at after the of the of LD-associated proteins during adipocyte differentiation from we staining of and at various after SVCs were after the showed a The of in perinuclear of and were with nascent LDs in the as as in SVCs at this LDs perilipin, or or in were LDs to be and than or and LDs grew to μm in on the of LDs, as In the of and it showed in the cytoplasm than on the of The temporal and spatial variations of and in SVCs during adipocyte differentiation are with in 3T3-L1 cells reported G. P. J. H. are associated with an of events in of a of the Full Text Full Text PDF PubMed Scopus Google Scholar). the of and that of of LD-associated proteins in we also staining of and at and after the as as at the shown in the of cells in cells from to images at each it found that and of cells at and the and of were also in the with on the of LDs in of and of cells at and In the of on LDs in of cells at the LDs were coated with The revealed that of the SVCs by after the and of the SVCs the of LD-associated proteins from to by after the We the between by the diameter of the in each and of the LD-associated proteins at The of LDs coated with and with and were found to be μm and μm in the of LDs coated with found to be μm in The revealed that the with of LD-associated the of LDs in SVCs is more than μm in the LDs are coated with as as However, the μm in the LDs are coated with are coated with that the SVCs have of LD-associated proteins from to In the observations and after the we on the adipocytes morphological that were to appear in the of the cell images of perilipin, and cell in such an adipocyte and after the In an by an in on the of LDs, the on the images that the of LD-associated proteins The most is that stained with in two of the suggesting that the adipocyte in a cell division the cell than other it to the cell and other cells in the other than that appear of in We also found that several adipocytes having LDs coated with or coated with and were in a cell division after the that adipocytes the of cell division before and even after the of LD-associated proteins never while SVCs were into adipocytes SVCs accumulating LDs, they to a and to on the The that is for the of adipocyte differentiation from SVCs derived from mesenteric adipose tissue previous in SVCs derived from epididymal and adipose tissues of (3Tikkanen M.J. Plant sterols and stanols. In Handbook of Experimental Pharmocology. Springer-Verlag, Berlin2005: 215-230Google Scholar, 4Bosner M.S. Lange L.G. Stenson W.F. Ostlund Jr., R.E. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry..J. Lipid Res. 1999; 40: 302-308Abstract Full Text Full Text PDF PubMed Google Scholar, 6Heinemann T. Axtmann G. von Bergmann K. Comparison of intestinal absorption of cholesterol with different plant sterols in man..Eur. J. Clin. Invest. 1993; 23: 827-831Crossref PubMed Scopus (348) Google Scholar) or in 3T3-L1 cells T.A. Tilvis R.S. Kesaniemi Y.A. Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population..Am. J. Epidemiol. 1990; 131: 20-31Crossref PubMed Scopus (556) Google Scholar). We that this is to of two Our time-lapse with a more of of primary and the that is be a specific characteristic of SVCs derived from mesenteric adipose tissue, although observations for SVCs derived from other tissues or 3T3-L1 cells are to be Numerous small LDs (a few micrometers in diameter) were in the perinuclear region at an early stage of adipocyte differentiation showed that nascent LDs (several in diameter) developed in the subsequently, of grew into small LDs while moving the perinuclear region. The of LDs are with in 3T3-L1 cells reported M. Plant sterol and and 2000; PubMed Google Scholar). a stage of adipocyte several small LDs grew to more than 10 μm in diameter while the of LDs According to these growth of LDs is by the of LDs with each In several nascent LDs appear to with each other of two adjacent LDs However, the LDs have to the of the LDs in the observation be to a between two We have developed a method for the fluorescence staining of LDs in living adipocytes In previous reports R.S. Miettinen T.A. Serum plant sterols and their to cholesterol J. Clin. Nutr. PubMed Scopus Google Scholar, H. M. J. J. M. R. Miettinen T.A. in the and with cholesterol absorption and Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar, K.E. von Bergmann K. Lutjohann D. R. Grundy S.M. Hobbs H.H. of sterols and to in and Lipid Res. 2002; Full Text Full Text PDF PubMed Google Scholar), LDs in adipocytes were stained with However, method cell and temporal and spatial variations of LDs can be in living adipocytes. We that the and of nascent LDs were in stained and adipocytes. However, fluorescence at for a the stained adipocytes in the region to a and from the that the cells were by the between the of fluorescence images fluorescence The most in the is that of SVCs cell division even after they LDs such cell division that the “final” cell division during adipocyte differentiation occurs considerably later than that characterized in 3T3-L1 cells. and the that SVCs by after the and that the of LD-associated proteins from to by after the The temporal of in SVCs during adipocyte differentiation is with in 3T3-L1 cells reported G. P. J. H. are associated with an of events in of a of the Full Text Full Text PDF PubMed Scopus Google Scholar). In 3T3-L1 the of LDs that the factor has been is a primary of Jr., R.E. J. Stenson W.F. absorption and of and in humans..Am. J. 2002; PubMed Scopus Google Scholar). The of reported to be to growth arrest M. and B. M. cell of activity of as reviewed in Ref. 10Patel S.B. Salen G. Hidaka H. Kwiterovich P.O. Stalenhoef A.F. Miettinen T.A. Grundy S.M. Lee M.H. Rubenstein J.S. Polymeropoulos M.H. et al.Mapping a gene involved in regulating dietary cholesterol absorption. The sitosterolemia locus is found at chromosome 2p21..J. Clin. Invest. 1998; 102: 1041-1044Crossref PubMed Scopus (157) Google Scholar). In we the of adipocyte division in time-lapse it that of the adipocyte divisions occurred after that after adipocytes of the adipocyte divisions occurred after that after LDs were coated with that SVCs were capable of division even after LDs were coated with that is to cell division of and that therefore the of has with that of cell We the between and the of cell with the of LD-associated the of with that of cell that has also with the of cell In in the and the of LDs between of adipocytes that during adipocyte differentiation and the that and growth of LDs and cell division, which in adipocyte are or than or the of primary SVCs is more than that of 3T3-L1 cells during adipocyte differentiation. In the of adipocyte division shown in LDs that in a adipocyte were evenly between two daughter cells or According to a previous (11Bjorkhem I. Boberg K.M. Leitersdorf E. Inborn errors in bile acid biosynthesis and storage of sterols other than cholesterol.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. In The Metabolic Bases of Inherited Disease. McGraw-Hill, New York2001: 2961-2988Google Scholar), primary unilocular adipocytes from adipose tissues can undergo division in of the two In cell division, a single into LDs, after which adipocytes divide and evenly distribute the LDs between two daughter cells. in cell division, a cell that has a small of LDs from a unilocular in other daughter cell a large, single The cell division of SVCs during adipocyte differentiation to the cell division of unilocular adipocytes than the cell In a previous D. and K. cell from mature adipocytes. Res. Scholar), it that primary unilocular adipocytes with LDs, that adipocyte on the other we never the morphological of adipocytes from a with LDs to a with In time-lapse observations have provided a detailed account of the morphological changes that SVCs undergo during adipocyte division and differentiation. We revealed temporal and spatial variations of LDs in individual cells during adipocyte and that adipocytes and that they LDs evenly between two daughter cells. Immunofluorescence observations of LD-associated proteins that the “final” cell division during adipocyte differentiation occurs considerably later than that characterized in 3T3-L1 cells. We also developed a method for the fluorescence staining of LDs in living adipocytes. observations using microscopy or fluorescence microscopy clarify on temporal and spatial variations of LDs, and they the between two LDs in living adipocytes. The are to and and for the and to for in by and for from the for the of with adipose lipid stromal-vascular cell