A novel technique to propagate primary human preadipocytes without loss of differentiation capacity

Objective: The study of human preadipocytes is hampered by the limited availability of adipose tissue and low yield of cell preparation. Proliferation of preadipocytes using common protocols, including fetal bovine serum (FBS), results in a markedly reduced differentiation capacity. Therefore, we were interested in developing an improved culture system that allows the proliferation of human preadipocytes without loss of differentiation capacity. Research Methods and Procedures: Adipose tissue samples were taken from subjects undergoing elective abdominal surgery. Cells were seeded at various densities and cultured using different formulations of proliferation media including factors such as fibroblast growth factor‐2 (basic fibroblast growth factor), epidermal growth factor, insulin, and FBS either alone or in combination. Cells were counted and induced to differentiate after confluence. After complete differentiation, cells were harvested, and glycerol‐3‐phosphate dehydrogenase activity was measured. Cells were subcultured for up to five passages. Results: The proliferation medium with 4 growth factors (PM4), consisting of 2.5% FBS, 10 ng/mL epidermal growth factor, 1 ng/mL basic fibroblast growth factor, and 8.7 µM insulin, resulted in lower doubling times at all seeding densities tested (0.05 × 10⁴ to 1.5 × 10⁴) compared with medium supplemented with 10% FBS. In contrast to cells in FBS medium, cells grown with PM4 medium retained full differentiation rate (glycerol‐3‐phosphate dehydrogenase activity, 493 ± 215 vs. 41 ± 17 mU/mg, p < 0.01). Differentiation capacity was fully retained at least for up to three passages in PM4 medium. Discussion: The use of PM4 medium results in substantial proliferation of human preadipocytes with preserved differentiation capacity. This novel technique represents a valuable tool for the study of human adipose tissue development and function starting from small samples.     

Adipocyte Development is Dependent Upon Stem Cell Recruitment and Proliferation of Preadipocytes

KRAS, KRYSTYNA M., DOROTHY B. HAUSMAN, GARY J. HAUSMAN, AND ROY J. MARTIN. Adipocyte development is dependent upon stem cell recruitment and proliferation of preadipocytes. Obes Res. Objectives: The ability to acquire fat cells persists over the life spans of animals. It is unknown whether adipocyte acquisition is the result of preadipocyte proliferation or stem cell recruitment to become adipocytes. The purposes of these studies were 1) to characterize early differentiation of stromal vascular (S-V) cells to preadipocytes as it is influenced by insulin, dexamethasone (DEX), and insulin-like growth factor-I (IGF-I); and 2) to determine whether new fat cells arise from stem cell recruitment or preadipocyte proliferation. Research Methods and Procedures: Freshly isolated S-V cells from rat inguinal adipose tissues were plated for 24 hours then exposed to serum-free medium. Results: Approximately 15% of freshly plated S-V cells were preadipocytes as determined by a preadipocyte specific marker, AD3. Total cell number and proportion of preadipocytes were significantly greater with 100 nM insulin treatment than with 0, 0. 1, or 1. 0 nM, but IGF-I treatment at 10 nM resulted in preadipocyte development similar to that with 100 nM insulin treatment. The addition of 5 nM DEX to the 100 nM insulin treatment resulted in a 20% increase in preadipocyte number by day 2 when compared to either treatment alone. 5-Bromo-2′-deoxyuridine treatment suppressed the increased proportion of preadipocytes from days 0–2 in non-insulin treated cells and prevented the increase typically observed with insulin. A mitosis inhibitor also significantly reduced the proportion of preadipocytes. Discussion: These results show for the first time that S-V cells are recruited as preadipocytes and that proliferation of these preadipocytes and early differentiation occur simultaneously.     

α-Poly-l-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes

α-Poly-l-lysine (PLL) has been used for various purposes such as cell attachment, immunization, and molecular delivery, and is known to be cytotoxic to several cell lines. Here, we studied the effect of PLL on the adipogenesis of 3T3-L1 cells and investigated the underlying mechanism. Differentiation media containing PLL with a molecular weight (MW) greater than 4 kDa enhanced lipid droplet formation and increased adipogenic marker levels, indicating an increase in adipocyte differentiation. PLL with a molecular weight between 30 and 70 kDa was more effective than PLL of other sizes in 3T3-L1 cell differentiation. Moreover, PLL induced 3T3-L1 adipogenesis in insulin-free adipocyte differentiation medium. Incubation with insulin and PLL exhibited greater adipogenesis than insulin treatment only even at a high concentration. PLL stimulated insulin signaling and augmented the signaling pathway when it was added with insulin. While PLL did not activate the glucocorticoid receptor, which is phosphorylated by dexamethasone (DEX), it showed a positive effect on the cAMP signal pathway when preadipocytes were treated with PLL and 3-isobutyl-1-methylxanthine (IBMX). Consistent with these results, incubation with PLL and DEX without IBMX induced adipocyte differentiation. We also observed that the mitotic clonal expansion phase was the critical stage in adipogenesis for inducing the effects of PLL. These results suggest that PLL functions as an adipogenic inducer in 3T3-L1 preadipocytes and PLL has a direct effect on insulin signaling, one of the main regulatory pathways.

     

Changes of pyridine nucleotide levels during adipocyte differentiation of mouse 3T3-L1 cells.

The levels of NAD and NADP were measured in 3T3-L1 cells during a differentiation from preadipocytes to adipocytes. The cells were grown in the ordinary medium and differentiated in the medium by adding dexamethasone, 1-methyl-3-isobutylxanthine, and insulin for 2 days, and then they were grown in the medium by adding only insulin for another 8 days to accumulate fat. The levels of cellular NAD and NADP increased abruptly with days after differentiation, and the levels of NAD and NADP reached maximum at day 7, and at day 10 the values were decreased compared with the maximum values. These results suggest that expression of the pyridine nucleotide biosynthesis genes is induced in the differentiation process.     

Adipocyte conversion of CHEF cells in serum-free medium

When grown in the presence of serum with added insulin, Chinese hamster embryonic fibroblasts (CHEF/18) cells can be induced to become preadipocytes that are committed to the adipocyte pathway of terminal differentiation (Sager, R., and P. Kovac, 1982, Proc. Natl. Acad. Sci. USA, 79:480-484). We found that commitment to the adipocyte pathway, as well as terminal differentiation to form mature adipocytes, can occur in a defined serum-free medium containing insulin. When CHEF/18 cells are plated in serum-containing medium, only 5-10% of cells in each colony undergo terminal differentiation, whereas in serum-free medium, greater than 90% of the cells became adipocytes. These and other results show that CHEF/18 cells require no adipogenic factors in addition to insulin and the other components of the serum-free medium (transferrin, epithelial growth factor, thrombin) to form adipocytes, and furthermore, that serum inhibits the rate of terminal adipocyte differentiation of these cells. As little as 10 ng/ml insulin added to serum-containing medium can induce adipogenesis, suggesting that insulin rather than an insulinlike growth factor is the active agent. The results further demonstrate that virtually every CHEF/18 cell can be induced into the adipocyte pathway.     

Changes in the nuclear triiodothyronine receptor during the differentiation of preadipocytic cell lines of mice

During the differentiation process of ob 17 preadipocytes in the presence of insulin, a progressive increase in the nuclear T3 receptor concentration per cell (1.5 to 3 fold) was observed. This increase was not found related to the addition of insulin to the culture medium and was concomitant to the expression of several phenotypes of the mature adipocyte. Triiodothyronine (T3) added to the culture medium of these cells increases the lipogenic pathway and several enzymes involved in fatty acid synthesis and esterification. After T3 addition at the same optimal physiologic concentration of 1.5 nM, the cellular concentration of its nuclear receptor was clearly reduced, without any significant alteration in the apparent affinity constant. A similar behavior was observed in 3T3-L1 preadipocytes. The molecular mechanism of action of the T3 nuclear receptor within the chromatin is not known. The biological relevance of its depletion by T3 itself remains to be determined.     

Differentiation of newborn rat preadipocytes in culture: effects of insulin and dexamethasone

A preadipocyte cell population isolated from the inguinal tissue of 3-day-old rats converts at confluence into mature adipocytes when cultured with insulin (10(-9) M). Insulin is necessary only from Day 4 postplating. If the addition of insulin is further delayed, the proportion of cells which will undergo adipose conversion decreases. A loss of the differentiation competence is also observed when the cells are allowed to proliferate (seeding at a low density in a serum containing medium). A preexposure of the primary cells to dexamethasone during the insulin-insensitive period (Days 0-4) accelerates the subsequent "insulin-dependent" adipose conversion. In order to produce its effect, dexamethasone needs only to be present for 4 h on Day 2 postplating. The effect of dexamethasone is probably due neither to inhibition of cell proliferation nor to induction of the cell content of insulin receptors. The evolution of G3PDH enzyme activity as well as of G3PDH protein and mRNA was used as an indicator of the differentiation process. The enzyme accumulates to a low extent during culture in the absence of insulin. When insulin is present, the enzyme level is dramatically increased (maximum on Day 11). Dexamethasone pretreatment (Days 0-4, or 4 h on Day 2) accelerated the G3PDH enzyme activity increase as well as protein and mRNA accumulation. This was also true in cells maintained in insulin-free medium; however, in this case, the increase in the enzyme activity was limited to the first 8 days of culture and full differentiation did not take place. We conclude that: (1) the rat preadipocytes are committed to differentiate, requiring insulin as a sufficient physiological stimulus; (2) the differentiation program is progressively lost after greater than 4 days of culture without insulin and more rapidly if the cells are allowed to undergo divisions; and (3) dexamethasone accelerates the insulin-dependent adipose conversion but alone does not ensure the complete differentiation process.     

Optimization of the differentiation of human preadipocytes in vitro

This study aimed at developing an optimal protocol for proliferation and differentiation of preadipocytes that is a prerequisite for constructing an ideal biohybrid composed of viable adipose precursor cells in a three-dimensional matrix. Such an implant could represent an adequate solution for correcting soft tissue defects, e.g., extensive deep burns or tumor resections. Preadipocytes were isolated from human subcutaneous adipose tissue samples and cultured in Dulbecco's modified eagle medium (DMEM)/Ham's F12 medium (F12) or OPTIMEM medium with or without the addition of human serum (hS) or fetal calf serum (FCS). The advantages of fibronectin-coated culture dishes for preadipocyte yield after isolation and differentiation were evaluated. After culture expansion, differentiation was induced by insulin, isobutylmethylxanthine, pioglitazone, dexamethasone, and transferrin in the absence of serum. The extent of differentiation was assayed by measuring the activity of glycerophosphate dehydrogenase as well as counting of differentiated versus undifferentiated cells. Our results show that fibronectin coating does not only strongly increase the yield of preadipocytes after isolation from adipose tissue but also significantly enhances differentiation of precursor cells to mature adipocytes. For optimal cell expansion, DMEM/F12 is more promoting than OPTIMEM and culturing with FCS shows a slightly better proliferation compared with hS supplementation. Differentiation, in contrast, is significantly improved when hS is used instead of FCS during proliferation. Our results smooth the way for autologous preadipocyte culturing and show that hS for preadipocyte culturing opens new and promising perspectives for adipose tissue engineering by optimizing in vitro expansion in cell culture and inducing substantial differentiation.     

Insulin and Insulin-like Growth Factor-1 Receptors Act as Ligand-specific Amplitude Modulators of a Common Pathway Regulating Gene Transcription

Insulin and insulin-like growth factor-1 (IGF-1) act on highly homologous receptors, yet in vivo elicit distinct effects on metabolism and growth. To investigate how the insulin and IGF-1 receptors exert specificity in their biological responses, we assessed their role in the regulation of gene expression using three experimental paradigms: 1) preadipocytes before and after differentiation into adipocytes that express both receptors, but at different ratios; 2) insulin receptor (IR) or IGF1R knock-out preadipocytes that only express the complimentary receptor; and 3) IR/IGF1R double knock-out (DKO) cells reconstituted with the IR, IGF1R, or both. In wild-type preadipocytes, which express predominantly IGF1R, microarray analysis revealed ∼500 IGF-1 regulated genes (p < 0.05). The largest of these were confirmed by quantitative PCR, which also revealed that insulin produced a similar effect, but with a smaller magnitude of response. After differentiation, when IR levels increase and IGF1R decrease, insulin became the dominant regulator of each of these genes. Measurement of the 50 most highly regulated genes by quantitative PCR did not reveal a single gene regulated uniquely via the IR or IGF1R using cells expressing exclusively IGF-1 or insulin receptors. Insulin and IGF-1 dose responses from 1 to 100 nm in WT, IRKO, IGFRKO, and DKO cells re-expressing IR, IGF1R, or both showed that insulin and IGF-1 produced effects in proportion to the concentration of ligand and the specific receptor on which they act. Thus, IR and IGF1R act as identical portals to the regulation of gene expression, with differences between insulin and IGF-1 effects due to a modulation of the amplitude of the signal created by the specific ligand-receptor interaction.     

Differentiation under the control of insulin of rat preadipocytes in primary culture: Isolation of homogeneous cellular fractions by gradient centrifugation

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10^?9 M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid:CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.     

The relationship between alkaline phosphatase activity and intracellular lipid accumulation in murine 3T3-L1 cells and human preadipocytes

Alkaline phosphatase (ALP) is expressed in 3T3-L1 preadipocytes, and its activity increases during adipogenesis. The purpose of this study was to determine whether ALP activity could be used as a measure of intracellular lipid accumulation in human preadipocytes and 3T3-L1 cells and which of the factors that induce adipogenesis are responsible for stimulating ALP activity. Adipogenesis was initiated in 3T3-L1 cells by incubation with differentiation medium containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. The effect of leaving out each of the differentiation medium components was studied. Adipogenesis was also assessed in human preadipocytes and 3T3-L1 cells in the presence of the ALP inhibitor histidine. ALP activity was measured using an automated colorimetric assay and intracellular lipid accumulation was measured using the lipid-specific dye oil red O. Removal of insulin or dexamethasone from the differentiation medium had little effect on either ALP activity or lipid accumulation in 3T3-L1 cells, while removal of IBMX blocked both. Histidine inhibited ALP activity and adipogenesis in human preadipocytes and 3T3-L1 cells. Pearson univariate correlation analysis demonstrated strong correlations between ALP activity and lipid accumulation in human preadipocytes (r=0.78, n=69) and in 3T3-L1 cells (r=0.92, n=27). These data suggest that ALP and fat storage are tightly linked during preadipocyte maturation and that the measurement of ALP activity may be a novel technique for the quantification of intracellular lipid accumulation that is more sensitive and rapid than currently used methods.     

Oleate promotes differentiation of chicken primary preadipocytes in?vitro

In addition to providing energy and constituting cell membrane, fatty acids also play an important role in adipocyte differentiation and lipid metabolism. As an important member of monounsaturated fatty acids, oleate, together with other components, is widely used to induce chicken preadipocyte differentiation. However, it is not clear whether oleate alone can induce chicken preadipocyte differentiation. In the present study, four different treatments were designed to test this question: basal medium, IDX [insulin, dexamethasone and IBMX (isobutylmethylxanthine)], oleate and IDX plus oleate. Cytoplasmic lipid droplet accumulation and mRNA expression for adipogenesis-related genes were monitored. After treatment of oleate on chicken preadipocytes, apparent lipid droplet formation and lipid accumulation were observed, accompanied by increasing expression of PPARγ (peroxisome proliferator-activated receptor-γ) and AFABP (adipocyte fatty acid-binding protein), but decreasing level of GATA2 (GATA-binding protein 2). In contrast, for cells cultured in the basal medium with or without IDX supplementation, lipid droplet barely occurred. These results suggest that exogenous oleate alone can act as an inducer of preadipocyte differentiation into adipocytes.     

Adipocyte Secreted Factors Enhance Aggressiveness of Prostate Carcinoma Cells

Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1) and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01). Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01). Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05). Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.     

Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

Acquisition of increased hormone sensitivity during in vitro adipocyte development.

Murine 3T3-L1 fibroblasts enter a differentiation program subsequent to prolonged maintenance in the confluent state and develop into adipocytes. The hormone sensitivity of adenylate cyclase and the physiological responsiveness to insulin were compared in 3T3-L1 preadipocytes and adipocytes. The following observations, comprising several distinct categories of hormone responsiveness, were made. (a) (2.5 micronM) isoproterenol stimulated adenylate cyclase 15-fold in adipocyte homogenates, but only 2.5-fold in preadipocyte preparations, suggesting a considerable magnification in beta-adrenergic responsiveness during development. (b) A totally new control element, adrenocorticotropic hormone responsiveness, was incorporated into the adenylate cyclase system of the adipocytes. (c) Sensitivity to prostaglandin E1 was observed in both preadipocytes and adipocytes, but no change in responsiveness could be detected in the differentiated cells. (d) Glucagon-sensitive adenylate cyclase could not be detected in either preadipocytes or adipocytes. (e) Both preadipocytes and adipocytes possess considerable insulin binding activity, but near physiological levels of insulin stimulate the conversion of glucose to CO2 and lipid only in the differentiated cells.     

Heterogeneity in the physiological states and pharmacological responses of differentiating 3T3-L1 preadipocytes

Increases in key components of adipogenesis and lipolysis pathways correlate at the population-averaged level during adipogenesis. However, differentiating preadipocytes are highly heterogeneous in cellular and lipid droplet (LD) morphologies, and the degree to which individual cells follow population-averaged trends is unclear. In this study, we analyze the molecular heterogeneity of differentiating 3T3-L1 preadipocytes using immunofluorescence microscopy. Unexpectedly, we only observe a small percentage of cells with high simultaneous expression of markers for adipogenesis (peroxisome proliferator-activated receptor γ [PPARγ], CCAAT/enhancer-binding protein α, and adiponectin) and lipid accumulation (hormone-sensitive lipase, perilipin A, and LDs). Instead, we identify subpopulations of cells with negatively correlated expressions of these readouts. Acute perturbation of adipocyte differentiation with PPARγ agonists, forskolin, and fatty acids induced subpopulation-specific effects, including redistribution of the percentage of cells in observed subpopulations and differential expression levels of PPARγ. Collectively, our results suggested that heterogeneity observed during 3T3-L1 adipogenesis reflects a dynamic mixture of subpopulations with distinct physiological states.     

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.