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.     

Direct and indirect effects of leptin on preadipocyte proliferation and differentiation

Leptin has been shown to reduce body fat in vivo. Adipocytes express the leptin receptor; therefore, it is realistic to expect a direct effect of leptin on adipocyte growth and metabolism. In vitro studies examining the effect of leptin on adipocyte metabolism require supraphysiological doses of the protein to see a decrease in lipogenesis or stimulation of lipolysis, implying an indirect action of leptin. It also is possible that leptin reduces adipose mass by inhibiting preadipocyte proliferation (increase in cell number) and/or differentiation (lipid filling). Thus we determined direct and indirect effects of leptin on preadipocyte proliferation and differentiation in vitro. We tested the effect of leptin (0-500 ng/ml), serum from leptin-infused rats (0.25% by volume), and adipose tissue-conditioned medium from leptin-infused rats (0-30% by volume) on preadipocyte proliferation and differentiation in a primary culture of cells from male Sprague-Dawley rat adipose tissue. Leptin (50 ng/ml) stimulated proliferation of preadipocytes (P<0.05), but 250 and 500 ng leptin/ml inhibited proliferation of both preadipocyte and stromal vascular cell fractions (P<0.01), as measured by [3H]thymidine incorporation. Serum from leptin-infused rats inhibited proliferation of the adipose and stromal vascular fractions (P=0.01), but adipose tissue-conditioned medium had no effect on proliferation of either cell fraction. None of the treatments changed preadipocyte differentiation as measured by sn-glycerophosphate dehydrogenase activity. These results suggest that leptin could inhibit preadipocyte proliferation by modifying release of a factor from tissue other than adipose tissue.     

Effects of fetal versus postnatal sera upon adipose tissue stromal-vascular cells in primary culture

Summary This experiment was conducted to determine if serum factors are responsible for differences in cellularity of prenatal and postnatal pig adipose tissue as determined by in vitro measurement of cellular proliferation and enzyme-histochemical metabolic development. Cellular proliferation of stromal-vascular cells derived from rat inguinal adipose tissue was measured by [³H]-thymidine incorporation. Coverslip cultures were used for analysis of histochemical differentiation. Cells were incubated in media containing 10% fetal bovine, fetal pig, mature pig, or various combinations of these sera. Fetal bovine serum promoted more [³H]-thymidine incorporation than fetal or postnatal pig sera. Fetal pig sera also stimulated more [³H]-thymidine incorporation than mature pig sera. Sera from adult pigs promoted differentiation and lipid filling of adipocytes. Fetal pig sera stimulated histochemical expression of enzymes, but did not induce lipid filling. Fetal bovine serum produced histochemically undifferentiated cells. Addition of fetal bovine serum to media containing mature pig sera reduced lipid accumulation and histochemical reactivity of cells. This effect of fetal serum was thus due to specific inhibition of lipid deposition and not substrate restriction. These experiments demonstrated that serum factors have a major influence on morphological development of fetal and postnatal adipose tissue.     

Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells

Stromal-vascular cells from the epididymal fat pad of 4-week-old rats, when cultured in a medium containing insulin or insulin-like growth factor, IGF-I, triiodothyronine and transferrin, were able to undergo adipose conversion. Over ninety percent of the cells accumulated lipid droplets and this proportion was reduced in serum-supplemented medium. The adipose conversion was assessed by the development of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, [14C]glucose incorporation into polar and neutral lipids, triacylglycerol accumulation and lipolysis in response to isoproterenol. Similar results were obtained with stromal-vascular cells from rat subcutaneous and retroperitoneal adipose tissues. Stromal-vascular cells required no adipogenic factors in addition to the components of the serum-free medium. Insulin was required within a physiological range of concentrations for the emergence of LPL and at higher concentrations for that of GPDH. When present at concentrations ranging from 2 to 50 nM, IGF-I was able to replace insulin for the expression of both LPL and GPDH. The development of a serum-free, chemically defined medium for the differentiation of diploid adipose precursor cells opens up the possibility of characterizing inhibitors or activators of the adipose conversion process.     

The relationship between serum and cell type on the development of rat and pig cultured preadipocytes

Stromal-vascular cells from rats and pigs were isolated from adipose tissue and used to measure preadipocyte proliferation and differentiation. Cells from rats and pigs were grown in either 2.5% pig serum or 2.5% rat serum. Cells were either supplemented or unsupplemented with insulin after five days of growth in culture. In these cultures, pig fat cells developed as discrete clusters while rat fat cells developed as loose clusters or as individual cells. Rat cells had greater levels of sn-glycerol phosphate dehydrogenase activity compared to pig cells. Rat serum increased soluble protein in plated cells when compared to cells grown in pig serum. Pig serum increased glycerol phosphate dehydrogenase specific activity when compared to rat serum. In this system, there was no response to insulin. The cells grown in rat serum did not resemble adipocytes in regard to the presence of large lipid droplets (oil red 0 staining). These results demonstrate that rat and pig stromal-vascular cells in culture are morphologically different. Cells from both species, however, responded similarly to sera from either species showing that cells from rats and pigs responded to the growth and differentiation factors present in these sera.Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Inhibition of growth hormone-stimulated lipolysis by somatostatin, insulin, and insulin-like growth factors (somatomedins) in vitro

The effects of somatostatin, insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II)/MSA on growth hormone (GH) (1 microgram/ml)-induced lipolysis were examined employing chicken adipose tissue in vitro. Basal and GH-stimulated glycerol release were inhibited by somatostatin (1 ng/ml) and by IGF-II/MSA (10 and 100 ng/ml). Insulin and IGF-I (10 and 100 ng/ml) completely inhibited the lipolytic response to GH without affecting basal glycerol release. Insulin and IGF-I were equipotent in inhibiting GH-induced lipolysis while IGF-II is only 16% as potent as insulin.     

Established cell lines from rat adipose tissue that secrete lipoprotein lipase

A number of cell lines derived from the stromal-vascular fraction of rat adipose tissue have been established that represent a variety of morphologic types. Despite their differing morphology, all of these cell lines secrete lipoprotein lipase in response to heparin. Because lipoprotein lipase secretion has been attributed to the presence of preadipocytes in the stromal-vascular fraction, we examined these cell lines for adipocyte conversion. None of the cell lines converted to adipocyte morphology when held at confluency or when exposed to media supplemented with high concentrations of fatty acid or very low density lipoproteins. These cell lines therefore do not seem to be preadipocytes, despite the presence of lipoprotein lipase. Among these cell lines are several that display the "cobblestone" morphology of endothelial cells, although they lack angiotensin-converting enzyme activity, reactivity with Factor VIII antibodies, and Weibel-Palade bodies. A number of authentic endothelial cells were found to be negative for lipoprotein lipase secretion. These data suggest that the "endothelial-like" cell lines established from adipose tissue are not endothelial cells.     

Growth hormone alters metabolic effects and proteolysis of insulin in adipose tissue during lactation.

To study the regulation of lipogenesis in adipose tissue by insulin and growth hormone during lactation, tissue was biopsied from primiparous bovines at 30 days antepartum and 60 days postpartum. Tissue was cultured for 24 hr or 48 hr in M199 with acetate and glucose, with a change of medium at 24 hr. The three in vitro treatments were: insulin and hydrocortisone at 10 and 50 ng/ml, respectively (IH); IH + 10 ng/ml of growth hormone (G10); and IH + 100 ng/ml of growth hormone (G100). IH allowed lipogenesis rates from 50% to 85% of those in fresh tissue. Addition of 10 ng/ml of growth hormone reduced (P less than 0.05) lipogenesis; at 100 ng/ml, the effect was only slightly greater. The hypothesis that insulin and growth hormone could be degraded by bovine adipose tissue was tested. Adipose tissue cell-free extracts degraded 125I-labeled insulin, but did not degrade labeled growth hormone. The insulin protease activity was further characterized and had a pH optimum of 7.1, a maximum hydrolysis of approximately 70%, and a hydrated molecular mass of approximately 23,000 daltons. Insulin proteolysis was inhibited by specific insulin protease inhibitors and stimulated by disulfide reducing agents. Bovine growth hormone, prolactin, and histone inhibited (P less than 0.05) the proteolysis of insulin, while bovine serum albumin, egg albumin, trypsin inhibitor, and lysozyme did not. Adipose tissue from pregnant and lactating bovines was sensitive to insulin and growth hormone, and growth hormone may modulate activity of an insulin-specific protease.     

Biochemical and cytochemical studies of preadipocyte differentiation in serum-free culture of porcine stromal-vascular cells: interaction of dexamethasone and growth hormone.

Stromal-vascular cells from adipose tissue of pigs 5-7 days of age were grown in serum for 2-3 days and switched to serum-free (insulin, transferrin and selenium) conditions +/- test hormones for 6-7 days. The interaction of dexamethasone (DEX) and human growth hormone (hGH) was evaluated since glucocorticoids augment and hGH antagonizes the effect of insulin. Low levels (1-10 nM) of DEX with insulin doubled (p less than 0.05) specific activity of glycerol phosphate dehydrogenase (GPDH) and doubled (p less than 0.05) the number of detectable fat cells relative to insulin alone. DEX with insulin enhanced the morphological differentiation of preadipocytes and markedly increased fat cell cluster numbers in the presence of hGH. Furthermore, 1-10 nM of DEX partially blocked (p greater than 0.05) the inhibitory effect of 10 nM hGH on GPDH activity, but 1-100 nM DEX had no effect (p greater than 0.05) on the ability of hGH to compromise lipid deposition. DEX alone (no insulin or hGH) induced the appearance of esterase-reactive but lipid-free cells. Cells with these characteristics were increased in number by DEX in the presence of hGH but were nearly absent in the presence of insulin and DEX. Therefore, transient exposure to GH in vivo may have no permanent effect on adipose tissue development in the continued presence of glucocorticoids.     

Human adipose tissue stromal vascular fraction cells differentiate depending on distinct types of media

Abstract.  Objectives : Angiogenesis, the process of formation of blood vessels, is essential for many physiological as well as pathological processes. It has been shown that human adipose tissue contains a population of non-characterized cells, called stromal-vascular fraction (SVF) cells, which are able to differentiate into several lineages. The aim of this study was to determine conditions for promoting differentiation of human adipose tissue progenitors towards endothelial cells, as well as to show that SVF cells cooperate with differentiated endothelium in capillary network formation. Materials and methods : Stromal vascular fraction cells were isolated according to modified Hauner's method and after adaptation they were cultured in pro-angiogenic or pro-adipogenic medium. Cells were characterized by presence of surface antigens by flow cytometry, and by expression of genes characteristic for endothelial cells or for adipocytes, quantitative real-time polymerase chain reaction. A number of tests were performed to verify their differentiation. Results : Differentiation of human SVF cells towards endothelium was stimulated by the presence of serum and absence of adipogenic factors, documented by the pattern of gene expression as well as different functional in vitro assays. SVF cells were found to work together with human umbilical vein endothelial cells to form capillary networks. Conclusions : Here, we show that differentiation of SVF cells to endothelial cells or adipocyte-like cells depended on the medium used. Our work provides a clear model for analysing the differentiation capacity of SVF cells.     

Extracellular matrix substrata alter adipocyte yield and lipogenesis in primary cultures of stromal-vascular cells from human adipose

The stromal-vascular fraction of human adipose was subjected to in vitro adipogenesis on different extracellular matrix substrata. Adipose tissue was harvested from the breast of 25 to 45 year-old female patients undergoing elective surgery. After 24 d, less than 5% of stromal-vascular cells had converted to adipocytes on fibronectin, 13% to 28% on tissue culture plastic and collagen I; and 59% +/- 7% on Matrigel. Lipid volume surpassed 4.5 x 10(3) microm3 cell(-1) for Matrigel and was 30% lower for the other substrata. Cell proliferation was evident for Matrigel and fibronectin, and cell spreading was most pronounced for fibronectin with a projected area exceeding 3 x 10(3) microm2 cell(-1). These results are relevant to the design of an adipose implant, providing insight into its feasibility and scaffold composition.     

Rat epididymal fat tissue express all components of the renin-angiotensin system

In the present study the gene expression of components of the renin-angiotensin system was investigated in fat tissue of rats. mRNAs for angiotensinogen, renin, angiotensin-converting enzyme and type I (AT1) angiotensin II receptor were detected in the stromal-vascular fraction of the fat tissue and the same mRNAs, with the exception of the angiotesin-converting enzyme, in the adipocyte fraction. Renin and angiotensin-converting enzyme activity was measured. The main source of renin activity was found in adipocytes and some minor activity in the stromal-vascular fraction, while the majority of the angiotensin-converting enzyme activity was in the stromal-vascular fraction. The present data provide evidence for the presence of the active renin-angiotensin system in rat adipose tissue.     

Differentiation-dependent expression of obese (ob) gene by preadipocytes and adipocytes in primary cultures of porcine stromal-vascular cells

The relationship between obese (ob) gene expression and preadipocyte differentiation was examined in primary cultures of porcine stromal-vascular (S-V) cells by Northern-blot analysis using a pig ob cDNA probe. Isolated adipocytes expressed high levels of ob gene, but S-V cells did not express the ob gene. Cultures were seeded with fetal bovine serum (FBS) plus dexamethasone (Dex) for 3 days followed by ITS (insulin 5 μg/ml, transferrin 5 μg/ml, and selenium 5 ng/ml) treatment for 6 days. Detectable levels of ob mRNA first appeared at day 1 with very low activity of glycerol phosphate dehydrogenase (GPDH). Levels of ob mRNA increased in parallel with preadipocyte number or GPDH activity at the later times in cultures. The depletion of preadipocytes by complement-mediated cytotoxicity at day 3 of culture resulted in markedly decreased ob mRNA expression. Immunocytochemical analysis showed that ob protein was localized in the cytosol of preadipocytes and adipocytes. These data indicated that the ob gene is expressed by preadipocytes and ob gene expression may be correlated with preadipocyte recruitment as well as fat cell size.     

The phosphodiesterase inhibitor IBMX suppresses TNF-alpha expression in human adipocyte precursor cells: a possible explanation for its adipogenic effect.

Tumor necrosis factor-alpha (TNF) is known to inhibit fat cell development in vitro and to be expressed in adipose tissue suggesting that it may act as an auto-/paracrine regulator of adipose tissue mass in vivo. We demonstrate here that endogenous TNF-mRNA expression of cultured human preadipocytes and adipocytes is suppressed by the unspecific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), which is frequently used to trigger the differentiation process. As assessed by the measurement of glycerophosphate dehydrogenase, IBMX stimulated the differentiation of human preadipocytes in a dose dependent manner up to threefold but remained ineffective when cells were simultaneously treated with 1 nM TNF. These results suggest that the adipogenic effect of IBMX is mediated by suppression of endogenous TNF production.     

Effect of transforming growth factor-beta on insulin-like growth factor 1- and dexamethasone-induced proliferation and differentiation in primary cultures of pig preadipocytes.

The purpose of this study was to examine the effects of a known inhibitor, transforming growth factor-beta1 (TGF-beta1) versus the known stimulators insulin-like growth factor-1 (IGF-1) and dexamethasone (DEX) on pig preadipocyte differentiation in serum and serum-free primary cultures. In cultures with serum, preadipocyte and nonpreadipocyte replication was increased (p < 0.02) by IGF-1 and by TGF-beta1 (p < 0.05; p < 0.001). IGF-1 (10 nM) enhanced preadipocyte differentiation (p < 0.05) in serum-supplemented (1% pig serum) cultures, whereas TGF-beta1 (15 pM) reduced preadipocyte differentiation (p < 0.01) in the presence and absence of IGF-1. Furthermore, GPDH (SN-glycerol-3-phosphate dehydrogenase) specific activity (marker that indicates differentiation) was decreased (p < 0.05) by adding TGF-beta1 to serum-free cultures, but TGF-beta1 had little effect in serum-supplemented cultures. DEX significantly enhanced GPDH activity and fat cell cluster number, whereas pretreatment with TGF-beta1 eliminated the DEX enhancement. We have shown for the first time that TGF-beta can decrease (p < 0.01) the cellular secretion of IGF-1 by pig adipose tissue cells and counter the effects of exogenous IGF-1. These studies indicate that TGF-beta1 may not inhibit adipocyte development in the initial growth phase, but may inhibit differentiation and/or hypertrophy (lipid filling) at a later stage of development.     

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.     

Tumor Necrosis Factor‐α Stimulates Cell Proliferation in Adipose Tissue‐Derived Stromal‐Vascular Cell Culture: Promotion of Adipose Tissue Expansion by Paracrine Growth Factors

Objective: Elevated levels of tumor necrosis factor‐α (TNF‐α) protein and mRNA have been reported in adipose tissue from obese humans and rodents. However, TNF‐α has catabolic and antiadipogenic effects on adipocytes. Addressing this paradox, we tested the hypothesis that paracrine levels of TNF‐α, alone or together with insulin‐like growth factor‐I (IGF‐I), support preadipocyte development. Research Methods and Procedures: Cultured stromal‐vascular cells from rat inguinal fat depots were exposed to serum‐free media containing insulin and 0.2 nM TNF‐α, 2.0 nM TNF‐α, or 0.2 nM TNF‐α + 1.0 nM IGF‐I at different times during 7 days of culture. Results: TNF‐α inhibited adipocyte differentiation as indicated by a reduction in both immunocytochemical reactivity for the preadipocyte‐specific antigen (AD3; early differentiation marker) and glycerol‐3‐phosphate dehydrogenase activity (late differentiation marker). Early exposure (Days 1 through 3 of culture) to 0.2 nM TNF‐α did not have a long term effect on inhibiting differentiation. Continuous exposure to 0.2 nM TNF‐α from Days 1 through 7 of culture resulted in a 75% increase in cell number from control. There was a synergistic effect of 0.2 nM TNF‐α + 1 nM IGF‐I on increasing cell number by Day 7 of culture to levels greater than those observed with either treatment applied alone. Discussion: These data suggest that paracrine levels (0.2 nM) of TNF‐α alone or in combination with IGF‐I may support adipose tissue development by increasing the total number of stromal‐vascular and/or uncommitted cells within the tissue. These cells may then be recruited to become preadipocytes or may alternatively serve as infrastructure to support adipose tissue growth.     

Hormonal Regulation of Leptin mRNA Expression and Preadipocyte Recruitment and Differentiation in Porcine Primary Cultures of S-V Cells

The hormonal regulation of leptin mRNA expression and the association between leptin expression and adipocyte differentiation were examined in primary cultures of porcine S-V cells with Northern blot and immunocytochemical analysis. Seeding for 3 days with fetal bovine serum (FBS) with varying levels of dexamethasone (Dex) increased levels of leptin mRNA in a dosedependent manner in parallel with increases in the proportion of preadipocytes (AD-3 positive cells; AD-3, a preadipocyte marker). Six-day treatment with 10 or 850 nM insulin after FBS+Dex treatment resulted in a similar increase in leptin mRNA expression and morphological differentiation. However, significantly lower levels of leptin mRNA and smaller fat cells were observed in cultures treated with 1 nM insulin or 10 nM insulin-like growth factor-I (IGF-I). Dex-induced increases in leptin mRNA levels and AD-3 cell numbers were blocked completely by the addition of transforming growth factor-β (TGF-β) to FBS+Dex-treated cultures. However TGF-β significantly increased fat cell size and leptin mRNA expression when added to ITS (insulin, 850 nM; transferrin, 5 μg/ml; and selenium, 5 ug/mL) treated cultures during the lipid-filling stage. When added with FBS+DEX for the first 3 days, growth hormone (GH) did not influence the Dex-induced increase in AD-3 cells and leptin mRNA expression, but GH reduced leptin mRNA levels when added with insulin for 6 days after FBS+Dex. These results demonstrated that regulation of leptin mRNA expression by Dex, insulin, IGF-I, TGF-β, and GH may be associated with changes in preadipocyte number and fat cell size.