Effects of lipid-related factors on adipocyte differentiation of bovine stromal-vascular cells in primary culture

The effects of several factors related to lipids on bovine adipocyte differentiation were investigated in primary culture. Adipocyte differentiation was assessed by development of glycerol-3-phosphate dehydrogenase (GPDH) activity and morphological observation. Addition of triglyceride mixture (Intralipid), caprylic acid and very low-, low- and high-density lipoproteins (VLDL, LDL and HDL) stimulated bovine preadipocyte differentiation in serum-free condition. Especially, VLDL strongly increased both cell protein contents and GPDH activity, suggesting that it stimulated both proliferation and differentiation of bovine preadipocytes. Under Intralipid-induced condition, differentiation of preadipocytes from subcutaneous adipose tissues was more evident than those from omental adipose tissues. However, such depot difference was not observed in medium supplemented with indomethacin, which is a peroxisome proliferator-activated receptor (PPAR) gamma agonist. This suggests that the differentiation capacity of bovine preadipocytes was different between depots and such difference is dependent on the ability to utilize lipids as endogenous PPARgamma ligands. Therefore, lipid metabolites have the stimulatory effects on bovine adipocyte differentiation in vitro, and lipoproteins, especially VLDL, may play an important role in development of bovine adipose tissues in vivo.     

The influence of insulin, triiodothyronine (T3) and insulin-like growth factor-I (IGF-1) on the differentiation of preadipocytes in serum-free cultures of pig stromal-vascular cells

Adipose tissue stromal-vascular (S-V) cells were obtained from 1-d-old pigs by enzymatic digestion and then grown to confluence (1 to 4 d) in 10% fetal bovine serum. Cultures then were shifted to serum-free media for 7 d. One and 50 nM insulin with transferrin (5 micrograms/ml) significantly increased (P less than .05) the cytodifferentiation of preadipocytes in serum-free cultures of S-V cells compared with control cultures (no hormones). Fifty nanomolar and 1 microM insulin with transferrin increased the specific activity of glycerol-phosphate dehydrogenase (GPDH) and the soluble protein levels of S-V cultures compared with control cultures. Ten nanomolar insulin-like growth factor-1 (IGF-1) increased (P less than .05) GPDH specific activity, soluble protein and preadipocyte cytodifferentiation compared with control cultures. Triiodothyronine (T3) supplementation (2 to 10 nM) increased (P less than .05) GPDH specific activity and increased lipid droplet size in preadipocytes compared with control cultures (no T3 but with insulin and transferrin). Cytodifferentiation of preadipocytes and soluble protein levels were not enhanced by T3 supplementation. Furthermore, T3 did not influence any parameters when added to S-V cultures that did not contain insulin. These data demonstrate that physiological levels of insulin or IGF-I can stimulate porcine preadipocyte differentiation in the absence of other hormones or growth factors.     

Responsiveness to adipogenic agents in stromal-vascular cultures derived from lean and preobese pig fetuses: an ontogeny study.

Primary cultures of stromal-vascular (S-V) cells from adipose tissue were used to evaluate characteristics of preadipocytes from lean and preobese fetuses at several ages (50, 75, and 110 d). In insulin-supplemented (1 microM) cultures (serum free) there was a significant age x fetal genotype interaction (P less than .01) for glycerol-phosphate dehydrogenase specific activity (GPDH); GPDH activity was genotype-dependent at 110 d (preobese greater than lean). The responses of S-V cultures (preadipocyte development) to 2% pig serum and to insulin (serum free) were similar. Main effects of genotype and age were significant (P less than .05) for protein levels in pig serum and insulin-treated cultures. There was a significant genotype x age (P less than .05) interaction for GPDH activity and protein levels in cultures treated with dexamethasone + 3-isobutyl-1-methylxanthine (DEX-IBMX). Treatment with DEX-IBMX induced more preadipocyte development in cultures from preobese fetuses than in cultures from lean fetuses at 110 d (P less than .05). The responsiveness of S-V cultures to DEX-IBMX (enhanced development) increased considerably between 50 and 75 d regardless of fetal genotype, but there was little response in cultures form 50-d fetuses. Preadipocyte development in lean and preobese fetuses diverged between 75 and 110 d, resulting in many more preadipocytes in preobese fetuses at 110 d. Therefore, S-V cells from preobese fetuses (late term) may be inherently more sensitive to adipogenic agents than S-V cells from lean fetuses.     

Optimization of in vitro conditions for bovine subcutaneous and intramuscular preadipocyte differentiation

The objective of these experiments was to develop an in vitro cell culture system for differentiation of bovine preadipocytes, which will permit examination of differences in differentiation between intramuscular (i.m.) and subcutaneous (s.c.) bovine preadipocytes. Stromal-vascular cells from bovine i.m. and s.c. adipose depots were isolated and cultured. Clonally derived s.c. preadipocytes were used to determine the ability of insulin, bovine serum lipids, octanoate, acetic acid, dexamethasone (DEX), and troglitazone (TRO) to elicit differentiation of these cells when added to serum-free medium. Addition of 10 and 20 microL/mL of a commercially available serum lipids supplement to low-glucose Dulbecco's modified Eagle's medium containing 280 nM insulin increased glycerol-3-phosphate dehydrogenase (GPDH) activity (P < 0.01). Inclusion of 1.25 to 10 microM TRO to medium containing 280 nM insulin and 20 microL/ mL serum lipids supplement also increased GPDH activity (P < 0.001) compared with 0 microM TRO. The combination of 280 nM insulin, 1 mM octanoate, and 10 mM acetic acid, with 48 h exposure to 0.25 microM DEX caused morphological differentiation in a small number of cells but did not stimulate GPDH activity (P = 0.99). When used together, 280 nM insulin, 20 microL/mL of serum lipids supplement, 40 microM TRO, and 0.25 microM DEX stimulated differentiation compared with the aforementioned treatment (P < 0.001). Omission of TRO or insulin from this medium reduced GPDH activity by 68% (P < 0.001), whereas removal of DEX tended to reduce GPDH activity (P = 0.06). Preadipocytes from s.c. (n = 3) and i.m. (n = 2) adipose tissues of 3 steers were used to determine the effects of TRO on differentiation using the established conditions. Forty to sixty microM TRO enhanced differentiation compared with 0 microM TRO (P < 0.02) in both depots. No depot differences in response to TRO were detected (P = 0.32). These data demonstrate that bovine preadipocytes are capable of differentiation in response to combinations of insulin, serum lipids, DEX, and TRO. Although TRO enhanced differentiation of bovine preadipocytes, no differential effects of TRO on the differentiation of s.c. and i.m. cells were detected.     

Porcine preadipocyte proliferation and differentiation: a role for leptin?

The present study was designed to determine whether porcine leptin can alter the proliferation and differentiation of the porcine preadipocyte. The stromal vascular cell fraction of neonatal pig s.c. adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. For differentiation studies, cells were seeded on six-well tissue culture plates and proliferated to confluency in 10% (vol/vol) fetal bovine serum (FBS) in Dulbecco's modified Eagle medium/F12 (DMEM/F12; 50:50). Cultures were differentiated using 2.5% pig serum (vol/vol) and recombinant porcine leptin at concentrations of 0 to 1,000 ng/mL alone or in combination with porcine insulin (100 nM), dexamethasone (1 microM), or IGF-1 (250 ng/mL). After 7 d of lipid filling, cultures were harvested for analysis of sn-glycerol 3 phosphate dehydrogenase (GPDH) and lipoprotein lipase (LPL). The GPDH and LPL activities are measures of preadipocyte differentiation. Data were corrected for protein content of the cultures. For proliferation experiments, 24 h after seeding cells with 10% FBS in DMEM/F12 in 25-cm2 tissue culture flasks, cells were switched to 5% FBS and supplemented with 0 to 1,000 ng of porcine leptin or 1,000 ng of murine leptin. Cell proliferation was measured by 3H-thymidine incorporation in preconfluent cultures over 24 h on d 4 of culture. At confluency, cells were switched to a medium to promote differentiation and lipid filling (2.5% pig serum, 100 nM insulin, 1 microM dexamethasone) for 7 d. Cells were harvested from the flasks and adipocytes were separated from stromal cells by Percoll gradient centrifugation. In a series of experiments, leptin alone or in combination with insulin, dexamethasone, or IGF-I did not affect differentiation as measured by the activity of GPDH and LPL. Leptin at any concentration did not inhibit differentiation induced by insulin, dexamethasone, or IGF-I; however, leptin at 1,000 ng/mL stimulated a 30% increase in preadipocyte proliferation (P = 0.007; n = 6) and a 27% increase in stromal cell proliferation (P < 0.001; n = 6). These results indicate that, at most, porcine leptin may contribute to the recruitment of new adipocytes within the adipose tissue.     

Differentiation of bovine intramuscular and subcutaneous stromal-vascular cells exposed to dexamethasone and troglitazone

The objectives of these experiments were to compare differentiation of bovine stromal-vascular (S-V) cells isolated from i.m. and s.c. adipose tissues in response to a glucocorticoid and a peroxisome proliferator-activated receptor gamma agonist. Stromal-vascular cells were isolated from i.m. and s.c. fat depots of 3 Angus steers and propagated in culture. Cells were exposed to differentiation media containing 0.25 microM dexamethasone (DEX), a glucocorticoid analog, and 40 microM troglitazone (TRO), a peroxisome proliferator-activated receptor gamma agonist, or both. Cells treated with DEX and TRO had greater (P < 0.02) glycerol-3-phosphate dehydrogenase activity than control cells. No interactions between DEX, TRO, and depot (P > 0.59) or depot differences (P = 0.41) in glycerol-3-phosphate dehydrogenase activity were found. Morphological assessment of adipogenic colonies showed that DEX induced a 1.8-fold increase in the percentage of adipogenic colonies (P = 0.03), whereas TRO increased the proportion of adipogenic colonies by 1.9-fold (P = 0.02) compared with those not treated with DEX or TRO, respectively. Depots had a similar percentage of adipogenic colonies (P = 0.18); however, the percentage of differentiated cells within adipogenic colonies was found to be 6.4-fold greater in s.c. isolates compared with i.m. (P < 0.001). Addition of TRO increased the proportion of differentiated cells within colonies by 10-fold compared with those of nontreated colonies (P < 0.001), whereas the percentage of differentiated cells within adipogenic colonies only tended to be increased by DEX (P = 0.10). These data indicate that bovine i.m. and s.c. S-V cells are capable of enhanced differentiation in response to DEX and TRO, and these effects were additive. Most importantly, inherent differences in the capacity to differentiate exist between adipogenic bovine i.m. and s.c. S-V cells.     

犬脂肪间充质干细胞的分离培养及分化潜能研究

为获得犬脂肪间充质干细胞,本试验取犬腹股沟皮下脂肪组织,分别利用组织培养法和酶消化法分离犬脂肪来源间充质干细胞,对比观察不同来源细胞的形态和增殖特征,并通过诱导液促进细胞向成骨细胞和成脂细胞方向分化,检测其分化潜能。结果表明,通过组织培养法培养的青年犬脂肪组织,可获得大量脂肪间充质干细胞,该细胞生长旺盛,形态均一,可分化为碱性磷酸酶染色阳性的成骨细胞和油红O染色阳性成脂细胞。组织培养法分离培养犬脂肪间充质干细胞操作简单,可为细胞移植治疗等研究提供充足的细胞来源。     

Culture of porcine stromal-vascular cells in serum-free medium: differential action of various hormonal agents on adipose conversion

We developed a strictly controlled serum-free culture system and tested the effects of adipogenic and antiadipogenic agents on the proliferation and(or) adipose conversion of porcine stromal-vascular cells. To avoid any interference with serum components, stromal-vascular cells were isolated, plated, and grown in absence of serum. In these culture conditions, a very limited growth phase and the absence of cell confluence were observed. However, when compared with continuous culture in serum-containing medium, the serum-free conditions were significantly more adipogenic as assessed by increased lipid content and increased enzymatic activities for lipoprotein lipase, glycerol 3-phosphate dehydrogenase, and malic enzyme. In serum-free medium, physiological concentrations of insulin or IGF-I were sufficient to significantly increase the percentage of lipid-containing cells, whereas triiodothyronine (T3) and GH had no effect. Insulin, IGF-I, and, more moderately, T3 also accelerated the lipid filling in the lipid-containing cells. In the presence of insulin, stimulation by T3 or hydrocortisone alone had no effect on glycerol 3-phosphate dehydrogenase activity, whereas their concomitant addition significantly increased it. Chronic exposure to tumor necrosis factor-alpha dose-dependently stimulated cell proliferation but clearly inhibited differentiation. Epidermal growth factor, another known antiadipogenic agent, also significantly increased the proliferation of stromal-vascular cells, but, surprisingly, this was not correlated with inhibition of adipocyte differentiation. Indeed, epidermal growth factor treatment did not decrease lipid filling and significantly improved lipoprotein lipase and malic enzyme activities. Taken together, the results obtained in these strictly controlled serum-free culture conditions point out functions for insulin, IGF-I, hydrocortisone, and T3 during early and(or) later steps of porcine adipose conversion. In addition, this study reports a positive activity of epidermal growth factor on porcine adipocyte differentiation that is in clear contrast with previous works performed with rodent cells.     

The influence of human growth hormone on preadipocyte development in serum-supplemented and serum-free cultures of stromal-vascular cells from pig adipose tissue

The influence of human growth hormone (hGH) on the differentiation of preadipocytes was examined in primary cultures of stromal-vascular (s-v) cells from porcine adipose tissue. In these experiments, cells were exposed to test media for 7–8 days after seeding and plating for two days in fetal bovine serum. In serum-free (insulin, transferrin and selenium) cultures hGH (1 and 10 nM) reduced the number and size of fat cell clusters (P<.05) by 50% relative to controls (no hGH). Differentiation of preadipocytes was assayed by labelling dividing cells with tritiated thymidine under identical conditions and then exposing cultures to test media for seven days. Fat cells were then separated from the other cells and radioactivity was determined in each fraction. In serum containing (2% pig serum) cultures hGH (10 nM) inhibited (P<.05) the differentiation of labelled preadipocytes. In cultures with serum and with 1 μM insulin and in serum-free cultures, 1 and 10 nM hGH reduced (P<.05) the levels of glycerol phosphate dehydrogenase (GPDH) specific activity by approximately 50%. However, hGH (1 and 10 nM) had no affect on GPDH activity in cultures with serum but without insulin. These studies indicate that hGH significantly impedes porcine preadipocyte development in vitro. Therefore, the decreased rate of adipose tissue growth observed in pigs chronically treated with GH could be due in part to impaired preadipocyte growth.     

A comparison of thiazolidinedione-induced adipogenesis and myogenesis in stromal-vascular cells from subcutaneous adipose tissue or semitendinosus muscle of postnatal pigs

This study compared the adipogenic potential of porcine stromal-vascular (S-V) cells from semitendinosus muscles and s.c. adipose tissue using thiazolidinediones. Stromal-vascular cells were obtained from s.c. adipose tissue and both semitendinosus muscles from 5- to 7-d-old pigs after collagenase digestion. Preadipocyte recruitment was measured using immunohistological evaluation for AD-3, a preadipocyte antibody. Ciglitazone increased the number of preadipocytes in adipose tissue but not semitendinosus muscle S-V cell cultures, whereas 10 microM troglitazone increased preadipocyte abundance in both adipose and muscle S-V cultures by approximately 3-fold (P < 0.05). Increasing troglitazone doses did not further increase preadipocyte number. Increases in preadipocytes were paralleled by increases in CCAAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) positive cells in adipose tissue S-V cultures, whereas PPARgamma-reactive but not C/EBPalpha-reactive cells were increased in muscle S-V cultures treated with 10 microM troglitazone. Additionally, troglitazone treatment did not increase lipid content in s.c. adipose tissue or muscle S-V cell cultures. Cells plated on laminin-precoated culture dishes were used to determine whether troglitazone influenced adipogenesis or myogenesis in cocultures from muscle S-V cells. There was no effect on the number of myotubes or the average number of nuclei per myotube, suggesting myogenesis was not impaired by troglitazone treatment. These results suggest that regulation of intramuscular adipogenesis differs from that of subcutaneous adipogenesis.     

Changes in adipocyte size during post-embryonic growth in the female Aylesbury duck

Mean adipocyte size was determined in inguinal, subcutaneous and peritoneal adipose tissues during the first 8 weeks of postembryonic growth. Increases in adipocyte volume are compared with the overall increases in adipose tissue mass during the same period.     

猪SGK家族基因的生物信息学分析及其在猪脂肪组织和细胞中的表达

【目的】 扩增猪血清和糖皮质激素诱导型激酶（SGK）家族基因并进行生物信息学分析,探索其在猪脂肪组织和细胞中的表达模式。【方法】 以藏猪脂肪细胞cDNA为模板PCR扩增SGK家族基因,通过在线工具预测其编码蛋白的理化性质及亚细胞定位;用Mega X软件构建系统进化树;采集30日龄巴马猪心脏、肝脏、脾脏、肾脏、肺脏、背肌、腿肌、颈部脂肪、背部脂肪、腹股沟脂肪、肾周脂肪等组织及7日龄和4月龄猪腹股沟脂肪组织,通过实时荧光定量PCR检测SGK家族基因在猪不同部位组织中的表达;采集30日龄巴马猪腹股沟脂肪组织并分离基质血管成分（SVF）细胞,诱导SVF细胞向白色脂肪细胞分化,通过实时荧光定量PCR检测SGK家族基因及脂肪分化标记基因CCAAT增强子结合蛋白α（C/EBPα）、过氧化物酶体增殖物激活受体（PPARγ）在脂肪细胞中的表达。【结果】 SGK1、SGK2和SGK3基因CDS区序列长度分别为1 296、1 104和1 473 bp,分别编码431、367和490个氨基酸;SGK1和SGK2定位于细胞质,SGK3定位于细胞核,三者均为亲水性蛋白,3个蛋白均含有相同基序,保守性高;系统进化树结果表明,猪与牛的亲缘关系最近;SGK1和SGK3基因在心脏、肝脏、脾脏、肺脏、肾脏、多种肌肉及脂肪组织广泛表达,SGK2基因在颈部、背部、腹股沟、肾周脂肪组织中均有较高表达;SGK1和SGK2基因在7日龄猪脂肪组织中表达量极显著高于4月龄猪脂肪组织（P<0.01）,SGK3基因在4月龄和7日龄的猪脂肪组织中表达量无显著差异（P>0.05）,且SGK3基因的表达量低于SGK1和SGK2基因;与未分化脂肪细胞相比,在分化后的脂肪细胞中SGK1和SGK2基因的表达量极显著上调（P<0.01）,且SGK1基因的表达量远高于SGK2基因,而SGK3基因的表达量无显著变化（P>0.05）。【结论】 SGK家族蛋白具有保守结构域,可能发挥着相似的功能,SGK1和SGK2可能参与调控猪脂肪细胞的分化过程,结果可为探究猪脂肪沉积的分子机制提供一定的理论基础。     

Effect of bone morphogenetic protein-4 (BMP-4) on adipocyte differentiation from mouse embryonic stem cells

Embryonic stem (ES) cells can differentiate spontaneously into various lineages in vitro. However, spontaneous commitment of ES cells to the adipocyte lineage is rare. In the present study, bone morphogenic protein-4 (BMP-4) is described as a factor inducing adipocyte differentiation from ES cells at a high rate. For this reason, ES-cell-derived embryoid bodies (EBs) in suspension cultures were exposed to different doses of BMP-4 for 5 days before they were plated onto gelatin-coated tissue culture plates. Moreover, the effect of serum-containing and serum-free media in three different combinations was assessed. Plated EBs, stained with Sudan Black and processed for transmission and scanning electron microscopy, were observed daily for adipocyte formation. Treatment with BMP-4 resulted in the appearance of adipocyte clusters in EBs' outgrowth, depending on the doses applied. Early in differentiation, many small fat droplets were observed in adipocytes, while later on they coalesced and formed a few large fat droplets. Adipocyte clusters had a fibrillar and vascular stroma, and each adipocyte was surrounded with a reticular external lamina. Furthermore, the appearance and development of adipocytes and their changes following 2-3 weeks of starvation mimicked live adipose tissue. In fact, understanding the biological activity of growth and differentiation factors is needed to regulate and direct stem cell differentiation to specific cell types in vitro.