Insulinlike growth factor-1 (IGF-1)-induced stimulation of porcine preadipocyte replication

Summary Insulinlike growth factor-1 (IGF-1) is both adipogenic and mitogenic to preadipose cell lines as well as primary stromal-vascular (SV) cells. The precise effect of IGF-1 on primary preadipocytes per se, however, has not been elucidated directly. In this study, primary porcine preadipocytes were exposed to IGF-1 while at three culture densities. The proportion of replicating preadipocytes was determined by labeling cells with an antiadipocyte/preadipocyte monoclonal antibody (MAb) concomitant with DNA measurement with propidium iodide. Flow cytometric analysis revealed that different seeding densities did not affect the relative proportion of preadipocytes (AD-1 positive) in cultures. However, IGF-1 treatment increased the proportion of preadipocytes at all densities but to a greater extent in more dense cultures. The resultant number of fat cell clusters formed was greater at higher densities and on IGF-1 treatment. The proportion of replicating cells in cultures decreased with increasing density. IGF-1 significantly increased replication at all densities and increased the number of replicating preadipocytes to the same extent independent of density. These results provide direct evidence of hormonal regulation of primary preadipocyte replication. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.     

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.     

Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells

Progress has been made in elucidating the cell-surface phenotype of primary adipose progenitors; however, specific functional markers and distinct molecular signatures of fat depot-specific preadipocytes have remained elusive. In this study, we label committed murine adipose progenitors through expression of GFP from the genetic locus for Zfp423, a gene controlling preadipocyte determination. Selection of GFP-expressing fibroblasts from either subcutaneous or visceral adipose-derived stromal vascular cultures isolates stably committed preadipocytes that undergo robust adipogenesis. Immunohistochemistry for Zfp423-driven GFP expression in?vivo confirms a perivascular origin of preadipocytes within both white and brown adipose tissues. Interestingly, a small subset of capillary endothelial cells within white and brown fat also express this marker, suggesting a contribution of specialized endothelial cells to the adipose lineage. Zfp423(GFP) mice represent a simple tool for the specific localization and isolation of molecularly defined preadipocytes from distinct adipose tissue depots.     

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.     

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 stroma-vascular fraction of rat inguinal and epididymal adipose tissue and the adipoconversion of fat cell precursors in primary culture

The stroma-vascular fraction (SVF) of inguinal and epididymal fat pads of 4 week-old rats was studied by electron microscopy. Among the various cell types, endothelial cells and preadipocytes were found in both SVF, while mesothelial cells were only detected in the epididymal SVF. The resulting heterogeneity of primary culture and the adipoconversion of the fat cell precursors were studied in a serum-supplemented medium enriched with insulin (14.5 nM) and exogenous triglycerides. Despite the heterogeneity of the inoculum, the primary cultures were rather homogeneous, fat cell precursors being the main cell type. Distinctive contaminant fibroblast-like cells were observed in both cultures, whereas epithelial-like cells, which correspond most probably to mesothelial cells, were only found in epididymal cultures. Differentiation of fat cell precursors was assessed by the appearance of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH). LPL activity was found in the same level in cells of both deposits while GPDH activity was elevated in inguinal vs epididymal derived stroma-vascular cells. The different adipose conversion pattern of both cultures was confirmed by morphological quantification: the maturation of epididymal fat cell precursors was faster but less extensive. These differences could be related mainly to regional localization rather than to different maturation of the two fat deposits.     

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.     

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.     

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.     

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 development of adipocytes in primary stromal-vascular culture of fetal pig adipose tissue

Primary cultures of stromal-vascular cells of adipose tissue from fetuses at 70 and 110 days of gestation were evaluated as potential model systems for studies of fetal adipocyte differentiation and proliferation. In the cultures, fat cells developed as very discrete clusters. Fat cell cluster development was dependent on initial cell density and time. Histochemical analysis for NADP-dependent dehydrogenases revealed an age of donor effect. Similar levels of enzymes (malate and glucose-6-phosphate dehydrogenase) were apparent in fat cell clusters and stromal cells in cultures of cells from fetuses at 70 days of gestation. These enzymes were only present in fat cell clusters in cultures of cells from fetuses at 110 days of gestation. The distribution of histochemically detectable esterase activity was dependent on the cell density at time of analysis. In areas of high cell density, esterase was restricted to fat cell clusters whereas, both stromal cells and fat cells were esterase reactive in areas of low cell density. Omitting PMS from the dehydrogenase media revealed differences in enzyme reactions of cells grown on collagen-coated and uncoated glass surfaces. These studies demonstrate that primary cultures of stromal-vascular cells from 110-day-old fetuses would be a useful system to identify factors involved in adipocyte proliferation and differentiation.     

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.     

Development of a human adipocyte synthetic polymer scaffold.

Because of the need for methods for successful transplantation of autologous fat, the differentiation of human preadipocytes on surgical mesh coated with various extracellular matrix components was investigated. Biopsy specimens of human adipose tissue were collected from seven different patients and were subjected to collagenase digestion and selective filtration, resulting in primary cultures of human preadipocytes. Fluortex monofilament-expanded polytetrafluoroethylene (52-/xm pore size) was as a template for coating with either human collagen, albumin, or fibronectin, followed by sodding with established cultures of human preadipocytes. Sodding efficiency on the different matrices was determined by trypsinization of attached cells at different time periods. Preadipocytes did not attach to uncoated polytetrafluoroethylene, but did attach to protein-coated mesh, and in a variable manner. Fibronectin-coating resulted in the highest efficiency of sodding, with differentiation of preadipocytes to adipocytes as assessed by scanning electron -microscopy and conventional Oil Red O staining. Similar results were achieved by using rat (n = 6) perirenal adipose tissue. This new method of adipocyte scaffolding may be used for improving soft-tissue augmentation and serving as a delivery system for growth factors important in wound healing.     

Impaired fat storage capacity in adipocyte precursors of I versus C57BL mice

I mouse strain displays adipocyte hypoplasia responsible for smaller fat pad size compared with C57BL mice. We investigated possible alterations in the proliferation and/or differentiation capacity of preadipocytes from the stroma-vascular fraction of adipose tissue in the I mouse strain. Control C57BL and I mice were studied at 8 weeks of age, and both adipose and stromal cells were isolated from epididymal and inguinal adipose tissue localizations. Results showed that the lower epididymal adipose mass in I mice was accompanied by a decrease in stromal cell number compared with C57BL mice. In inguinal fat pads, total cell number in the stroma-vascular fraction was unmodified; lipoprotein lipase activity significantly increased in stromal cells from I mice compared with control mice. In this depot, further characterization of cells from the stroma-vascular fraction by separation of cells according to density showed an increased number of preadipocytes in the I mouse whole stromal cell population. These preadipocytes seemed unable to undergo terminal maturation, thus leading to a decrease in the number of mature adipocytes. These results indicated that resistance to fat accumulation in I mice is characterized by site-dependent impairment of both the proliferative rate and the differentiation capacity of adipocyte precursors.     

Glucocorticoid-induced androgen inactivation by aldo-keto reductase 1C2 promotes adipogenesis in human preadipocytes

Adipogenesis and lipid storage in human adipose tissue are inhibited by androgens such as DHT. Inactivation of DHT to 3α-diol is stimulated by glucocorticoids in human preadipocytes. We sought to characterize glucocorticoid-induced androgen inactivation in human preadipocytes and to establish its role in the antiadipogenic action of DHT. Subcutaneous and omental primary preadipocyte cultures were established from fat samples obtained in subjects undergoing abdominal surgeries. Inactivation of DHT to 3α/β-diol for 24 h was measured in dexamethasone- or vehicle-treated cells. Specific downregulation of aldo-keto reductase 1C (AKR1C) enzymes in human preadipocytes was achieved using RNA interference. In whole adipose tissue sample, cortisol production was positively correlated with androgen inactivation in both subcutaneous and omental adipose tissue (P < 0.05). Maximal dexamethasone (1 μM) stimulation of DHT inactivation was higher in omental compared with subcutaneous fat from men as well as subcutaneous and omental fat from women (P < 0.05). A significant positive correlation was observed between BMI and maximal dexamethasone-induced DHT inactivation rates in subcutaneous and omental adipose tissue of men and women (r = 0.24, n = 26, P < 0.01). siRNA-induced downregulation of AKR1C2, but not AKR1C1 or AKR1C3, significantly reduced basal and glucocorticoid-induced androgen inactivation rates (P < 0.05). The inhibitory action of DHT on preadipocyte differentiation was potentiated following AKR1C2 but not AKR1C1 or AKR1C3 downregulation. Specifically, lipid accumulation, G3PDH activity, and FABP4 mRNA expression in differentiated preadipocytes exposed to DHT were reduced further upon AKR1C2 siRNA transfection. We conclude that glucocorticoid-induced androgen inactivation is mediated by AKR1C2 and is particularly effective in omental preadipocytes of obese men. The interplay between glucocorticoids and AKR1C2-dependent androgen inactivation may locally modulate adipogenesis and lipid accumulation in a depot-specific manner.     

促酰化蛋白(ASP)诱导3T3-L1前脂肪细胞分化

促酰化蛋白 (ASP)代替经典激素“鸡尾酒”诱导法中胰岛素 ,通过形态学观察、油红染色分化百分比测定、脂肪细胞甘油三酯合成率和甘油三酯总量测定 ,并与经典激素“鸡尾酒”法诱导前脂肪细胞分化情况比较 ,探讨ASP是否具有诱导 3T3 L1前脂肪细胞分化作用 .ASP组诱导分化第 6d ,3T3 L1前脂肪细胞变大、变圆 ,出现大量脂肪滴 ,形态由前脂肪细胞向成熟脂肪细胞转变 ;随着诱导分化时间延长 ,胞浆中脂滴进一步积累 .分化 9d时 ,3T3 L1前脂肪细胞分化完全 .油红染色结果显示 ,ASP组分化率很高 (85 % ) ,与胰岛素组分化率 (90 % )相似 ,明显高于IBMX +DEX组 (4 0 % ) .ASP不仅促进 3T3 L1前脂肪细胞形态向成熟脂肪细胞转化 ,同时促进细胞中甘油三酯的合成和积累 .ASP组诱导分化第 3d时 ,脂肪细胞甘油三酯合成率明显高于对照组和IBMX +DEX组 ,但仍低于胰岛素组 ;在分化第 6d和第 9d时 ,ASP组甘油三酯合成率进一步升高 ,与对照组和IBMX +DEX组相比差异有极显著性 ,与胰岛素组相比无显著性差异 .ASP组诱导分化 3d时 ,脂肪细胞中甘油三酯总量明显高于对照组和IBMX +DEX组 ;分化 6d和 9d时 ,甘油三酯总量进一步升高 ,与对照组和IBMX +DEX组相比差异有极显著性 ,而与胰岛素组相比无显著性差异 .结果表明 ,新型脂源性激     

Expression of Masticatory-specific Isoforms of Myosin Heavy-chain,Myosin-binding Protein-C and Tropomyosin in Muscle Fibers and Satellite Cell Cultures of Cat Masticatory Muscle

We test the hypothesis that cat jaw satellite cells belong to a distinct lineage preprogrammed to express masticatory-specific isoforms of myosin heavy-chain (m-MyHC), myosin-binding protein-C (m-MBP-C), and tropomyosin (m-Tm) during myogenesis in vitro. A monoclonal antibody (MAb) against m-MyHC and MAbs raised here against cat m-MBP-C and m-Tm were used to stain cryostat sections of cat masseter muscle and cultured myotubes derived from satellite cells of cat temporalis and limb muscles, using peroxidase immunohistochemistry. MAbs against m-MBP-C bound purified m-MBP-C in Western blots. MAbs against m-Tm failed to react with m-Tm in Western blots, but reacted with native m-Tm in gel electrophoresis–derived ELISA. In cat masseter sections, MAbs against m-MyHC, m-MBP-C, and m-Tm stained all masticatory fibers, but not the jaw-slow fibers. Cat jaw and limb muscle cultures mature significantly more slowly relative to rodent cultures. However, at 3 weeks, all three MAbs extensively stained temporalis myotubes, whereas they apparently stained isolated myotubes weakly in cat limb and rat jaw cultures. We conclude that satellite cells of masticatory fibers are preprogrammed to express these isoforms during myogenesis in vitro. These results consolidate the notion that masticatory and limb muscle allotypes are distinct. (J Histochem Cytochem 58:623–634, 2010)