Hormone-induced differentiation of agranular endoplasmic reticulum in the interstitial cells of the mouse testis

Summary The administration of chorionic gonadotrophin to prepuberal mice results in precocious maturation of the testicular interstitial cells. The cytoplasm of the nine-day-old cells is characterized by abundant lipid droplets, large numbers of glycogen particles and mitochondria. By contrast, the membranous organelles are poorly developed.Human chorionic gonadotrophin brings about mobilization of lipid droplets and glycogen particles, and differentiation of large areas of agranular endoplasmic reticulum.The present observations are in agreement with the reports that human chorionic gonadotrophin increases the secretion of testosterone and that the agranular endoplasmic reticulum is the site of storage of steroid and of the enzymes involved in the biosynthesis of androgens.     

Development of brown fat cells in monolayer culture. II. Ultrastructural characterization of precursors, differentiating adipocytes and their mitochondria

The stroma of mature brown fat has been shown to contain cells which can proliferate and accumulate fat in monolayer cultures, and which have inherent characteristics distinct from those of white fat precursor cells. The purpose of the present investigation was to characterize by electron microscopic analysis these brown fat cells and their subsequent development when they were grown in vitro. By comparison with the existing ultrastructural data on brown fat in situ, it could thus be determined whether or not the precursor cells have the capacity to differentiate in culture. The stromal-vascular fraction isolated from the brown fat of weaned rats was identified as containing adipocyte stem cells, preadipocytes, endothelial cells and a few mature adipocytes. During the first week in culture (i.e., growth phase to confluence), when multilocular fat accumulation occurred, the mitochondria of the preadipocytes developed cristae and matrix granules, as they do in differentiating brown fat in situ. Such granules have been shown to be a sign of intense inner membrane synthetic activity. After confluence, the mitochondria regressed in internal structure and became morphologically more similar to white fat mitochondria. It was concluded that mature brown fat contains precursor cells which can differentiate in vitro. However, this differentiation was incomplete, and the necessity of specific factors for a full mitochondrial development in brown fat is discussed.     

Programming human pluripotent stem cells into white and brown adipocytes

The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice, the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease.     

Histogenesis of human extraparenchymal Leydig cells

From 64 consecutive autopsies of patients with neither testicular nor hormonal pathology, 26 showed extraparenchymal Leydig cells, located mainly in the epididymis and in the spermatic cord. The ultrastructural study of these specimens plus those obtained from 2 patients affected with functional testicular tumors leads to the following conclusions: (1) The origin of ectopic Leydig cells is not interstitial Leydig cells having infiltrated the testicular nerves and migrated along them towards ectopic locations. (2) The ectopic Leydig cells are considered to develop from undifferentiated precursor cells, located extraparenchymally, mainly inside and beside the testicular nerves. These precursor cells are similar to those observed in the testicular interstitium and have an ovoid shape and some cytoplasmic projections. The cytoplasm contains vesicles of smooth endoplasmic reticulum, lysosomes, lipid droplets and abundant microfilament bundles. The transformation from these cells into mature Leydig cells implies a progressive differentiation of the cytoplasmic components involved in steroid biosynthesis.     

Seeing the trees in the forest: selective electroporation of adipocytes within adipose tissue

Electroporation has been recently adapted for the transfer of macromolecules into cells of tissues in vivo. Although mature adipocytes constitute <20% of cells residing in adipose tissue, we hypothesized that fat cells might be susceptible to selective electrotransfer of plasmid DNA owing to their large size relative to other cells in the tissue. Results demonstrate the feasibility of electroporating DNA into mature fat cells with >99% selectivity over other cells in the tissue. Further experiments used the "adiporation" technique to image the subcellular targeting of fluorescent bioreporter molecules to the nucleus, mitochondria, and lipid droplets of adipocytes within intact adipose tissue. Finally, we utilized fluorescent bioreporters to examine the effects of constitutive activation of the beta-adrenergic signaling pathway in adipocytes. These results demonstrate that overexpression of rat beta1-adrenergic receptors alters the cellular morphology of white adipocytes in a fashion that mimics the effects of systemic infusion of beta3-adrenergic receptor agonists. Hallmarks of the altered morphology include pronounced fragmentation of the single lipid droplet, repositioning of the nucleus, and induction of mitochondrial biogenesis. These results indicate that activation of beta-adrenergic signaling within adipocytes is sufficient to induce a phenotype that resembles typical brown adipocytes and suggest that in vivo electroporation will allow molecular dissection of the mechanisms involved.     

Movement of lipolytic products to mitochondria in brown adipose tissue of young rats: an electron microscope study

Lipolysis occurred and lamellar structures with a periodicity of 40 A developed in glutaraldehyde-fixed brown adipose tissue of suckling rats when the tissue was incubated at 25 degrees C. The lamellar structures were found in capillaries, associated with chylomicrons, in intracellular channels of capillary endothelium, in extracellular space, and in channels near lipid droplets in adipocytes in tissue of fed rats injected intravenously with chylomicrons. They were also found in channels near mitochondria and inside mitochondria in adipocytes in incubated-fixed tissue of rats exposed to 4 degrees C for 2 hr or unsuckled overnight. In addition, aqueous spaces developed adjacent to lipid droplets in incubated tissue of cold-exposed and unsuckled rats. Development of lamellar structures under conditions causing lipolysis and accumulation of fatty acids in fixed tissue indicated the lamellae were composed primarily of fatty acids. We conclude that fatty acids formed by lipolysis of chylomicrons in tissue from fed rats accumulated in a continuum of the outer leaflets of cell membranes extending from capillary lumen to lipid droplets of adipocytes, and fatty acids formed by lipolysis of intracellular lipid in tissue from cold-exposed or unsuckled rats accumulated mostly in a continuum extending from lipid droplets to the interior of mitochondria. When fatty acids overcrowded the continuum in fixed tissue, they formed lamellar extensions of the continuum at different sites along its course through the tissue.     

Immunohistochemical identification of the uncoupling protein in rat brown adipose tissue

Brown adipose tissue mitochondria are characterized by the presence of an uncoupling protein that gives them an exceptional capacity for substrate-controlled respiration and thermogenesis. The specific localization of this protein in rat brown adipocytes was demonstrated using an immunohistochemical technique, the peroxidase-antiperoxidase (PAP) method. Light microscopy observations showed that serum antibodies raised against the uncoupling protein selectively reacted with multilocular brown adipocytes. No labeling could be detected in either unilocular adipocytes, capillaries, or muscle fibers (striated and vascular smooth muscle). Staining was more intensive in certain adipocytes than in others, suggesting the presence of cellular heterogeneity. The specificity of the staining technique was demonstrated by showing that treatment of the preparations with antiserum saturated with an excess of uncoupling protein almost entirely inhibited brown adipocyte labeling. The specificity and selectivity of the PAP method allow the clear differentiation of uncoupling protein-containing adipocytes from other cellular types, suggesting that this immunohistochemical technique will represent an extremely useful tool for studying adipocyte function and differentiation.     

Ultrastructural analysis of the in vitro differentiation of female rat preadipocytes

In an attempt to characterize the preadipocytes of the adipose tissue of female rat, we studied by electron microscopy the differentiation of the cells into mature adipocytes in in vitro cultures. The preadipocytes arose from the stroma-vascular fraction of perirenal and perigenital adipose tissue. Culture of the preadipocytes in an enriched medium consisting of Dulbecco's medium supplemented with 10% fetal calf serum, antibiotics, rat triglycerides (0.5%), insulin (290 nM) and Tween 80 (0.1 mg/ml) induced their adipose conversion. The morphology of preadipocytes changed progressively. They accumulated fat granules, droplets and finally globules, which fused together. The cell organelles featured qualitative and quantitative modifications. The nucleus migrated with most mitochondria and a part of the Golgi system towards the cell periphery; the rough endoplasmic reticulum, dilated at the initial stage of differentiation became less and less conspicuous; the perinuclear Golgi system was dispersed between lipid droplets during fat accumulation; thick bundles of microfilaments, localized beneath the plasma membrane disappeared; large lipid droplets were surrounded by a network of microfilaments; many microvesicles and some "rosettes" typical of mature adipocytes could be observed. Nevertheless, the ultrastructural criteria did not allow to clearly discriminate the undifferentiated cells: early preadipocytes (without lipid droplets), adipoblasts and fibroblasts, all of these being probably present in the culture system.     

THE DIFFERENTIATION OF WHITE ADIPOSE CELLS : An Electron Microscope Study

Differentiating white adipose tissue from presumptive and developing fat pads of newborn and young rats was fixed in buffered osmium tetroxide, embedded in Vestopal W, and examined in an electron microscope. Pre-adipose cells were found to be fibroblasts characterized by their spindle shape, long tenuous cytoplasmic extensions, and profuse endoplasmic reticulum. The developmental stages traced from fibroblast to mature adipose cell show a gradual change in cell shape, an accumulation of cytoplasm and non-membrane-bounded lipid, a decrease in the endoplasmic reticulum, and a change in shape of mitochondria. Transitory glycogen appears at mid-differentiation. Numerous smooth-membraned vesicles occur in the cytoplasm throughout differentiation. Pinocytosis is constantly evident. Cells of the multilocular stage are shown to differ from brown fat cells, particularly with respect to cytoplasmic membrane systems and mitochondria. No transport of particulate lipid from the lumen of the capillary to, or within, the adipose cell was detected, nor could any cell organelle be demonstrated to be visibly related to lipid synthesis and/or deposition.     

Effects of noradrenaline exposure on rat brown adipocytes in cultures. An ultrastructural study

Adipocytes in intact brown adipose tissue show multivacuolar lipid deposit and mitochondria of 'typical' morphology. Cultured brown adipocytes retain the multivacuolar lipid deposit, while 'typical' mitochondria degenerate and 'atypical' organelles appear instead of the former. Since evidence exists that catecholamines deeply influence brown adipose tissue morphology and function in vivo, we undertook the present ultrastructural investigation to assess whether exposure of cultured brown fat cell to noradrenaline could prevent (or induce regression of) the in vitro morphological modifications of brown adipocytes. Brown adipocytes cultured for 8 h in the presence of noradrenaline (5 X 10(-5) M) had a larger mitochondrial area (i.e. a larger percentage of cytoplasm occupied by non-degenerating mitochondria) in comparison with control cells, as assessed by morphometry; this was due to larger number of mitochondria in noradrenaline-treated cells. Moreover, a number of cells with mitochondria strictly resembling those of the intact tissue were visible in noradrenaline-treated cultured after 8 hr, while 'typical' mitochondria were no longer observed in parallel control cultures. After 5 days of culture without hormone addition, exposure to noradrenaline (5 X 10(-5) M) did not induce quantitative modifications of 'atypical' mitochondria or changes of their ultrastructure up to 12 hr. However, reduction in size of the lipid deposit and activation of both rough endoplasmic reticulum and Golgi apparatus were evident in noradrenaline-treated adipocytes in comparison with non-treated cells.     

Ultrastructural studies on the pheromone-producing cells in the silkmoth, Bombyx mori: formation of cytoplasmic lipid droplets before adult eclosion

In Bombyx mori, pheromone-producing cells accumulate a number of lipid droplets in the cytoplasm preceding the production of the sex pheromone, bombykol. The process of lipid droplet formation in the pheromone-producing cells was investigated by using light and electron microscopy. Light microscopy revealed that the lipid droplets appeared from 2 days before adult eclosion and dramatic accumulation took place between 2 days and 1 day before eclosion. Electron microscopical studies revealed that smooth endoplasmic reticulum and numerous vesicles, their sizes being less than 1 microm, were detectable 2 days before eclosion, and some vesicles were fused with mitochondria at this stage. These characteristic changes in the pheromone-producing cells suggest that fatty acyl-CoA synthesis following de novo fatty acid synthesis takes place at this time. Involutions in the basal plasma membrane of the cells occurred throughout the observed period, which were extensive on the day before adult eclosion. Besides extensive basal involutions, immature lipid droplets appeared and then mature fully electron-dense lipid droplets were observed on the day of adult eclosion. These ultrastructural observations, combined with recent physiological studies suggest, that the basal involutions presumably reflect the uptake of lipidic components required for the construction of lipid droplets, the function of which is to store the bombykol precursor and to provide it for bombykol biosynthesis in response to pheromonotropic stimuli by pheromone biosynthesis activating neuropeptide (PBAN).     

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 microm 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.     

Recruitment of brown fat and conversion of white into brown adipocytes: Strategies to fight the metabolic complications of obesity?

The role of white and brown adipose tissues in energy metabolism is well established. However, the existence of brown fat in adult humans was until very recently a matter of debate, and the molecular mechanisms underlying brown adipocyte development remained largely unknown. In 2009, several studies brought direct evidence for functional brown adipose tissue in adults. New factors involved in brown fat cell differentiation have been identified. Moreover, work on the origin of fat cells took an unexpected path with the recognition of different populations of brown fat cell precursors according to the anatomical location of the fat depots: a precursor common to skeletal muscle cells and brown adipocytes from brown fat depots, and a progenitor cell common to white adipocytes and brown adipocytes that appear in certain conditions in white fat depots. There is also mounting evidence that mature white adipocytes, including human fat cells, can be converted into brown fat-like adipocytes, and that the typical fatty acid storage phenotype of white adipocyte can be altered towards a fat utilization phenotype. These data open up new opportunities for the development of drugs for obesity and its metabolic and cardiovascular complications.     

Cyclic AMP effects on cell-to-cell junctional membrane permeability during adipocyte differentiation of 3T3-L1 fibroblasts

Mouse 3T3-L1 fibroblast cells, also know as preadipocytes, differentiate in vitro into adipocytes when treated with promoting agents and acquire numerous properties characteristic of mature fat cells. We studied junctional cell-to-cell communication by measuring the incidence of electrical coupling and transfer of carboxy- fluorescein among these cells. When 3T3-L1 cells were induced to differentiate into adipocytes, they lost virtually all cell-cell communication. Preadipocytes that remained nondifferentiated after the treatment maintained normal communication. Loss of communication in the adipocytes invariably coincided with appearance of lipid droplets and not with other phenotypic changes. In the differentiating cells, loss of cell-to-cell communication and lipid accumulation was prevented if dibutyryl cyclic AMP and caffeine were present in the culture medium. Addition of dibutyryl cyclic AMP and caffeine to already differentiated adipocytes resulted in loss of lipid and simultaneously improved junctional permeability. The results demonstrate that in the in vitro 3T3-L1 cell system, (a) cell-to-cell communication and lipid synthesis are intimately related during the adipose conversion and (b) cAMP affects the expression of the two phenotypes.     

Microsomal Triglyceride Transfer Protein (MTP) Associates with Cytosolic Lipid Droplets in 3T3-L1 Adipocytes

Lipid droplets are intracellular energy storage organelles composed of a hydrophobic core of neutral lipid, surrounded by a monolayer of phospholipid and a diverse array of proteins. The function of the vast majority of these proteins with regard to the formation and/or turnover of lipid droplets is unknown. Our laboratory was the first to report that microsomal triglyceride transfer protein (MTP), a lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins, was expressed in adipose tissue of humans and mice. In addition, our studies suggested that MTP was associated with lipid droplets in both brown and white fat. Our observations led us to hypothesize that MTP plays a key role in lipid droplet formation and/or turnover. The objective of these studies was to gain insight into the function of MTP in adipocytes. Using molecular, biochemical, and morphologic approaches we have shown: 1) MTP protein levels increase nearly five-fold as 3T3-L1 cells differentiate into adipocytes. 2) As 3T3-L1 cells undergo differentiation, MTP moves from the juxtanuclear region of the cell to the surface of lipid droplets. MTP and perilipin 2, a major lipid droplet surface protein, are found on the same droplets; however, MTP does not co-localize with perilipin 2. 3) Inhibition of MTP activity has no effect on the movement of triglyceride out of the cell either as a lipid complex or via lipolysis. 4) MTP is found associated with lipid droplets within hepatocytes from human fatty livers, suggesting that association of MTP with lipid droplets is not restricted to adipocytes. In summary, our data demonstrate that MTP is a lipid droplet-associated protein. Its location on the surface of the droplet in adipocytes and hepatocytes, coupled with its known function as a lipid transfer protein and its increased expression during adipocyte differentiation suggest a role in lipid droplet biology.     

Differentiation-related changes in mitochondrial properties as indicators of stem cell competence

Several methods may be used to assess stem cell competence, including the expression of cell surface markers and telomerase activity. We hypothesized that mitochondrial characteristics might be an additional and reliable way to verify stem cell competence. In a multipotent, adult monkey stromal stem cell line, previously shown to differentiate into adipocytes, chondrocytes, and osteocytes, we found that several mitochondrial properties change with increasing passage number in culture. Cells from the earliest passage (P11) versus those from a later passage (P17) are characterized by: (a) a much higher percentage of cells (85% vs. 18%) with a perinuclear arrangement of mitochondria; (b) a much lower percentage of cells (1% vs. 57%) with an aggregated mitochondrial arrangement, in which mitochondria appear to coalesce into large clumps; (c) a much lower percentage of cells with lipid droplets (1% vs. 36%), suggesting less differentiation into adipocytes; (d) a 5.6-fold lower ATP content per cell (0.45 vs. 2.51 pmoles ATP/cell; and (e) a 10-fold higher rate of oxygen consumption (37.8 vs. 3.8 nmoles O2/min/10(3) cells), indicating a higher metabolic activity. Collectively, these data indicate that the perinuclear arrangement of mitochondria, accompanied by a low ATP/cell content and a high rate of oxygen consumption, may be valid indicators of stem cell differentiation competence, while departures from this profile indicate that cells are differentiating or perhaps becoming senescent. These results represent the first characterization of mitochondrial properties reported for a primate stem cell line.     

Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also markedly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation.