Recruitment and differentiation of intramuscular preadipocytes in stromal-vascular cell cultures derived from neonatal pig semitendinosus muscles

The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte recruitment and expression of CCAAT/enhancing binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) proteins in stromal-vascular (SV) cell cultures derived from neonatal subcutaneous adipose tissue and semitendinosus muscles. One adipose tissue SV cell culture and one semitendinosus muscle SV cell culture were established from each of six young pigs (5 to 7 d of age). Conventional SV cell-culture procedures were used to digest adipose and muscle tissue and to harvest and culture adipose and muscle SV cells. Muscles were digested after the removal of all visible connective tissue from the excised muscle. One hour after seeding, muscle SV cell cultures were rinsed and refed new media to remove debris and insoluble muscle protein. The SV cell cultures were double-stained for lipid and the AD-3 antibody, a preadipocyte marker, at 1, 3, and 6 d and were double-stained for lipid and C/EBPalpha or PPARgamma at d 6. Preadipocytes were randomly distributed and not clustered after 1 d in muscle and adipose SV cultures. Regardless of treatment, relative and absolute fat cell numbers were lower (P < 0.05) in muscle than in adipose-SV cell cultures. The DEX treatments produced similar magnitudes of increase in relative and absolute preadipocytes and adipocytes in muscle- and adipose-SV cultures. Several extracellular matrix substrata had no influence on adipogenesis in muscle-SV cell cultures. These studies indicate that muscle-SV cultures are characterized by a low number of adipocytes under basal conditions and a low number of glucocorticoid-responsive preadipocytes.     

The influence of dexamethasone and insulin on expression of CCAAT/enhancer binding protein isoforms during preadipocyte differentiation in porcine stromal-vascular cell cultures: evidence for very early expression of C/EBPalpha

Expression of CAAT/enhancer binding protein (C/EBP) isoforms was examined in primary cultures of adipose tissue stromal vascular (S-V) cells before and during preadipocyte differentiation. Immunocytochemistry showed that the proportions and numbers of C/EBPalpha-, C/EBPbeta-, and C/EBPdelta-reactive cells were maximized after seeding and plating from d 0 to 3 in fetal bovine serum (FBS). However, there were few preadipocytes (AD-3+) and fewer cells with lipid and the number of C/EBPalpha-reactive cells clearly exceeded the number of preadipocytes. Seeding and plating in dexamethasone (DEX) + FBS from d 0 to 3 markedly increased the proportions and numbers of preadipocytes and C/EBPalpha-reactive cells compared to seeding and plating in FBS, d 0 to 3. The number of C/EBPalpha- and C/EBPbeta-reactive cells and preadipocyte reactivity for C/EBPbeta decreased with insulin or DEX treatment, d 3 to 6, following FBS, d 0 to 3. However, insulin + DEX treatment, d 3 to 6, maintained the number of C/EBPalpha-reactive cells and either maintained or increased preadipocyte reactivity for C/EBPalpha and C/EBPbeta. DEX and DEX + insulin treatment induced recruitment of a similar number of preadipocytes, but preadipocytes were not reactive for C/EBPalpha and C/EBPbeta in DEX-treated cultures. The number of C/EBPdelta reactive cells did not change from d 3 to 6 and was not influenced by hormone treatment. After DEX + FBS, d 0 to 3, the high numbers of C/EBPalpha-reactive cells and preadipocytes were maintained by insulin treatment alone. Western blot analysis for C/EBPalpha confirmed the immunocytochemical results. Double staining demonstrated that expression of C/EBPalpha protein was maximized before or at the onset of lipid accretion, whereas expression of C/EBPbeta protein was correlated with lipid accretion. These results indicate that coupling or integration of preadipocyte recruitment with C/EBPalpha expression may be a critical step in glucocorticoid-induced adipogenesis.     

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.     

Meat Science and Muscle Biology Symposium: the influence of extracellular matrix on intramuscular and extramuscular adipogenesis

The extracellular matrix (ECM) and specific ECM components can have a major influence on cell growth, development, and phenotype. The influence of the ECM and ECM components on adipogenesis in vivo and in vitro will be reviewed in this paper. Engelbreth-Holm-Swarm substratum and laminin per se markedly increased attachment, spreading, and hypertrophy of preadipocytes in serum-free primary cultures of porcine adipose tissue stromal-vascular cells. Furthermore, primary cultures of stromal-vascular cells showed that preadipocytes express ECM components after preadipocyte recruitment. Staining for plant lectins, type IV collagen, and laminin in fetal pig adipose tissue demonstrates that adipocyte reactivity for laminin was strong throughout fetal development and was similar for developing adipocytes and vasculature. However, lectin binding and type IV collagen reactivity of blood vessels preceded that for adipocytes. Therefore, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development. Specific inhibitors and modulators of collagen synthesis have been used to evaluate the role of collagens in the differentiation of bovine intramuscular preadipocytes (BIP) and other preadipocyte cell lines. Triglyceride accretion of BIP cells was inhibited by a general inhibitor of collagen biosynthesis, whereas specific inhibitors or modulators of type IV collagen inhibited 3T3-L1 cell differentiation. Further study revealed that compared with collagens types I to IV, type V and VI collagens have an important and active role in BIP adipogenesis. The growth of intramuscular bovine adipose tissue may be dependent on collagen newly synthesized and organized by the adipocytes per se. The role of extracellular or ECM proteolysis in regulating adipogenesis also will be reviewed in this paper. Many members of the matrix metalloproteinase (MMP) family are expressed by adipocytes, and specific inhibition of MMP-9 greatly reduces adipogenesis in vitro. Possibly, MMP and other proteases regulate turnover of key adipocyte ECM proteins that are involved in the regulation of preadipocyte proliferation and differentiation.     

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.     

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.     

A method to establish co-cultures of myotubes and preadipocytes from collagenase digested neonatal pig semitendinosus muscles

The relationships between adipocyte and muscle cell development within muscle are important in the study of factors or agents that may improve meat quality. Neonatal porcine muscle has the potential to yield both cell types for cell culture because it contains developing adipocytes and a high number of muscle satellite cells. Therefore, we modified a conventional collagenase-based procedure to digest neonatal porcine muscle and subsequently cultured the resultant muscle stromal-vascular (SV) cells on several substrata in basal and dexamethasone (DEX)-containing media. Developing myotubes and preadipocytes were present in muscle SV cell cultures on laminin substrata following seeding and plating with fetal bovine serum (FBS) with or without DEX. Myotube number was much higher (P < 0.05) on laminin substrata compared with all other substrata, whereas preadipocyte number in muscle SV cell cultures was independent of substrata, as we have shown previously. This approach can be used to establish co-cultures of differentiating adipocytes and myotubes from collagenase-digested neonatal pig muscle. Because the comparison is within the same culture dish, this method allows for a direct comparison of the responses of adipogenic and myogenic cells to growth and differentiation factors. For example, DEX did not alter myogenesis (i.e., 11 +/- 3 vs. 11 +/- 4 myotubes per unit area for control and DEX-treated cultures, respectively), but it has been shown to markedly increase preadipocyte number in muscle SV cell cultures.     

Feeding lambs high-oleate or high-linoleate safflower seeds differentially influences carcass fatty acid composition

Our objective was to determine effects of dietary high-oleate (Oleate; 76% 18:1) or high-linoleate (Linoleate; 78% 18:2) safflower seeds on fatty acids in muscle and adipose tissue of feedlot lambs. White-faced ewe lambs (n = 36) were fed a beet pulp, oat hay, and soybean meal basal diet (Control), blocked by BW, and allotted randomly to dietary treatments. Cracked safflower seeds were used in isocaloric and isonitrogenous replacement of beet pulp, oat hay, and soybean meal so that Oleate and Linoleate diets contained 5.0% additional fat. Fatty acids were determined in semitendinosus, longissimus dorsi (longissimus), and adipose tissue from the tail head (tailhead adipose tissue), adjacent to the 12th rib (s.c. adipose tissue), and kidney and pelvic fat (KPH adipose tissue) depots. Fatty acid data were analyzed within muscle and adipose tissue as a split-block design. Single degree of freedom orthogonal contrasts were used to compare treatment effects. Average daily gain, feed efficiency, and carcass characteristics did not differ (P = 0.15 to 0.96) across dietary treatments. Adipose tissue saturated fatty acids were greater (P = 0.04) for Controls but were not different (P = 0.36) in muscle. Trans-vaccenic acid (18:1(trans-11)) increased (P < 0.0001) with safflower supplementation and was greater (P < 0.0001) in Linoleate than in Oleate for both tissue types. Linoleate lamb had greater (P < 0.0001) PUFA than Oleate lamb in muscle and adipose tissue. Conjugated linoleic acids (CLA; cis-9, trans-11 and trans-10, cis-12) were greater (P < 0.0001) in muscle and adipose tissue of lambs fed safflower seeds. Lambs fed Linoleate had greater (P < 0.0001) CLA in adipose tissue and muscle than lambs fed Oleate. Saturated fatty acids were greater (P < 0.0001) in s.c. adipose tissue than in tailhead adipose tissue. Mono- and polyunsaturated fatty acids were greater (P < 0.0001) in tailhead adipose tissue than in s.c. adipose tissue. Weight percentages of 18:1(trans-11) ranked tailhead adipose tissue = KPH adipose tissue > s.c. adipose tissue and semitendinosus > longissimus, whereas CLA ranked tailhead adipose tissue > s.c. adipose tissue > KPH adipose tissue and semitendinosus > longissimus. Feeding mono- and polyunsaturated fatty acids increased tissue 18:1(trans-11) and CLA, which is a favorable change in regard to current human dietary guidelines.     

m6A甲基化酶METTL3和WTAP在牦牛组织及脂肪细胞中的表达

本研究旨在探究m⁶A RNA甲基化酶METTL3和WTAP在牦牛(Bos grunniens)不同组织、前体脂肪细胞增殖与分化过程中的表达模式和前体脂肪细胞分化过程mRNA m⁶A的变化水平。采用qRT-PCR检测牦牛皮下脂肪、肌肉、心、肝、脾、肺、肾和皮下脂肪不同时期(18和30月龄)METTL3及WTAP的mRNA表达水平。应用I型胶原酶消化法获取牦牛前体脂肪细胞,油红O染色和脂肪分化标志基因的检测建立牦牛前体脂肪细胞分化模型,以及qRTPCR检测前体脂肪细胞增殖分化阶段METTL3和WTAP的mRNA表达水平。结果表明,肝脏组织中METTL3表达最高(P<0.05),皮下脂肪组织表达量最低(P<0.05);WTAP在皮下脂肪组织中的表达最为丰富,脾脏中的表达量最低(P<0.05)。30月龄皮下脂肪组织中METTL3和WTAP mRNA表达量高于18月龄。前体脂肪细胞诱导分化12 d时,细胞中出现多而密的脂环,脂肪细胞分化特异性标志基因FABP4、C/EBPα和PPARγ第12天的表达量显著高于第0天(P<0.05)。METTL3和WTAP的表达量在细胞增殖阶段(24、48和72 h)呈现“下降-上升”的表达趋势(P<0.05)。在牦牛前体脂肪细胞分化阶段(0、4、8和12 d),METTL3表达量呈现“上升-下降-上升”的趋势,WTAP呈现“上升-下降”的趋势。细胞分化阶段mRNA m⁶A水平呈现逐渐上升的趋势,在分化12 d时细胞内RNA的m⁶A丰度最高(P<0.05)。本研究获得METTL3和WTAP在牦牛不同组织和前体脂肪细胞增殖分化阶段的变化规律及细胞分化过程中mRNA m⁶A水平变化,初步揭示WTAP和METTL3对牦牛脂代谢具有重要的调控作用。     

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.     

Expression of leptin mRNA and CCAAT-enhancer binding proteins in response to insulin deprivation during preadipocyte differentiation in primary cultures of porcine stromal-vascular cells

The aim of this study was to examine the correlation between CCAAT-enhancer binding proteins (C/EBPs) and leptin gene expression in response to insulin deprivation in preadipocytes and adipocytes. Adipose tissue from 7 d-old pigs was digested enzymatically and stromal-vascular (S-V) cells were seeded and plated for 3 d in fetal bovine serum (FBS) with dexamethasone (DEX) followed by 6 d (Days 3–9) in serum-free medium with insulin (850 nM or 10 nM), transferrin, and selenium. During FBS+DEX treatment (Days 0–3) a large number of preadipocytes develop with no lipid accretion. In contrast, preadipocyte number does not change with lipid accretion during insulin treatment (Days 3–9). Total RNA and cells were harvested from S-V cultures after periods with and without insulin after FBS+DEX. Northern-blotting and Western blot analysis were used to study leptin mRNA and C/EBP protein expression in cultures, respectively. Insulin deprivation from Days 3–4 reduced leptin mRNA and C/EBP- protein expression. Treatment with 850 nM or 10 nM insulin from Days 3–9 induced leptin mRNA and C/EBP- expression at a similar level. In cultures treated with 10 nM insulin from Days 3–7, leptin and C/EBP- expression were reduced markedly by insulin deprivation from Days 7–9, but were restored by insulin treatment for 6 hr before harvesting. The restoration of leptin expression by insulin was blocked by cycloheximide treatment. However, C/EBP-β protein levels did not change regardless of insulin deprivation. Insulin deprivation from Days 7–9 in cultures treated with 850 nM insulin from Days 3–7 did not influence C/EBP- or leptin mRNA expression, whereas C/EBP- and leptin expression were reduced after treating these cultures with 1.5 uM okadaic acid for 45 min before harvesting on Day 9. However, cycloheximide treatment for 6 hr before harvesting did not reduce leptin mRNA expression. These results suggest that 1) leptin expression is positively correlated with C/EBP- expression, and 2) the maintenance of leptin expression after insulin deprivation in 850 nM insulin-treated cultures on Day 9 may be associated with the presence of C/EBP- expression and/or activation.     

Glucocorticoids and the differentiation of porcine preadipocytes

The function of glucocorticoids in the differentiation of porcine preadipocytes was examined. Stromal-vascular cell cultures (containing preadipocytes) derived from adipose tissue of the perirenal, ham and shoulder regions of neonatal pigs were incubated in the presence of hydrocortisone at 0 to 100 ng/ml medium. Perirenal cells did not respond to hydrocortisone with an increase in enzyme expression, nor did they demonstrate growth characteristics similar to those of cultures derived from the ham or shoulder. Cultures from the shoulder and ham regions demonstrated dose-responsive increases in enzymatic expression to hydrocortisone. Enzymatic responses by cultures derived from the ham region were lower than responses by cultures from the shoulder region as measured by changes in the activities of sn-glycerol-3-phosphate dehydrogenase and lipoprotein lipase. Addition of insulin to the medium did not produce a synergistic effect with glucocorticoid on differentiation as determined by these enzymatic parameters. However, [14C]glucose metabolism by the cells in culture was synergistically increased by insulin and glucocorticoid supplementation of the medium. The ability of hydrocortisone to induce differentiation of porcine preadipocytes in vitro suggests that the changes that occur in plasma glucocorticoid concentrations during late gestation may play an important role in the rapid development of s.c. adipose tissue in the fetal pig. Secondly, the differences in culture characteristics and hormone responses of cells derived from different locations of adipose tissue formation indicate that differences may exist in the regulation of the growth and development of preadipocytes from different anatomical locations.     

Insulin-like growth factor 1 induction of differentiation of porcine preadipocytes

The role of insulin-like growth factor I (IGF-1) in the development of the porcine preadipocyte was studied. Primary cultures of stromal-vascular cells (containing preadipocytes) were derived from s.c. adipose tissue of pigs at 1 d of age by enzyme digestion and centrifugation. Cells were cultured for a total of 15 d. Cells were exposed to IGF-1 at concentrations of 0, 5, 25 or 50 ng/ml medium during one of four time periods: d 1-15, d 1-5, d 13-15, or 4 h on d 15 of culture. IGF-1 had a mitogenic effect on cells during the first three time periods as determined by coulter counting. IGF-1 induced the enzymatic differentiation of porcine preadipocytes following exposure for either the entire 15 d of culture or for only 48 h (d 13-15) after confluency had been attained (d 5). Histochemically, lipid accumulation over time paralleled changes in enzyme activity. Incubation of IGF-1 with the cell cultures during the logarithmic phase of growth (d 1-5) or for 4 h on d 15 did not affect enzyme activity. These data indicate that IGF-1 can induce the differentiation of porcine preadipocytes after the cells leave the logarithmic phase of growth through action on post-confluent events.     

Adipose tissue development in the fetal pig examined using monoclonal antibodies

Two anti-adipocyte monoclonal antibodies (MAbs: AD-1 and AD-2) have been used to study the development of dorsal s.c. adipose tissue in fetuses from 50 to 110 d of gestation. Immunofluorescent staining of cryostat sections with each antibody revealed antigen-positive cells in fetal s.c. mesenchyme prior to lipid deposition. Lipid droplets as well as AD-1 and AD-2 positive cells were detected within the underlying muscle at 50 d. From 70 to 110 d of development, the AD-1 and AD-2 MAbs each detected all adipocytes examined, as well as capillaries associated with fat cell clusters in s.c. tissues. Reactivity toward both antibodies, as well as lipid deposition, also was detectable in the muscle underlying the s.c. mesenchyme from 70 d onward. Each MAb possessed a distinct pattern of reactivity. The AD-2 MAb stained arrector pili muscles and vessels in the s.c. mesenchyme and vessels in the underlying muscles, whereas the AD-1 did not. No reactivity using either MAb was detectable toward any other cell types within s.c. tissues. These results established the presence of cells expressing surface determinants found on mature adipocytes and associated capillaries prior to adipogenesis. A lineage relationship between adipocytes and capillary endothelial cells is suggested.     

Differentiation of stromal-vascular cells isolated from canine adipose tissues in primary culture

A culture condition supporting adipocyte differentiation of stromal-vascular (S-V) cells isolated from canine adipose tissues was established. Morphological observation and determination of glycerol-3-phosphate dehydrogenase (GPDH) activity were used as the criteria for adipocyte differentiation. After reaching confluence, the cells were able to undergo terminal adipocyte differentiation by treatment with 100 microM indomethacin, 10 microg/ml insulin and 0.5 mM 1-methyl-3-isobutylxanthine (MIX) in medium supplemented with 5% fetal calf serum (FCS). In the absence of either indomethacin or insulin, the S-V cells did not undergo adipose conversion and GPDH activity was not increased, indicating that both indomethacin and insulin play essential roles in this culture system. The S-V cells from inguinal adipose tissues exhibited the greatest increase in GPDH activity among the four depots (inguinal > abdominal-subcutaneous > perirenal > omental). demonstrating that adipocyte differentiation was also intensely dependent on anatomic sites from which the S-V cells were derived. Interestingly, dimethylsulfoxide (DMSO) was found to accelerate adipocyte differentiation in combination with indomethacin and insulin. Under this condition, up to 90% of the cells displayed adipocyte phenotypes and the GPDH activity reached 1288 +/- 441 mU/mg protein. This culture system may be useful for investigating other adipogenic factors as well as anti-adipogenic factors involved in the regulation of canine adipose tissue development.     

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.     

Fasting regulates the expression of adiponectin receptors in young growing pigs

Adiponectin is an adipocyte-derived hormone that can improve insulin sensitivity. Its functions in regulating glucose utilization and fatty acid metabolism in mammals are mediated by 2 subtypes of adiponectin receptors (AdipoR1 and AdipoR2). This study was conducted to determine the effect of fasting on the expression of adiponectin and its receptors. The expression of adiponectin was not affected in s.c. adipose tissue, but adiponectin expression increased in visceral adipose tissue after fasting. In contrast, expression of both AdipoR mRNA was increased in the liver and s.c. adipose tissue of 24-h-fasted pigs compared with fed pigs, but the mRNA in muscle and visceral adipose tissue was not affected by fasting. A third putative adiponectin receptor, T-cadherin, was cloned and the mRNA expression was determined. T-Cadherin has been recognized to act as a vascular adiponectin receptor in vascular endothelial and smooth muscle cells. Our data showed that the expression of T-cadherin was decreased in the muscle of fasted pigs, suggesting that the expression of T-cadherin can be regulated by feeding status. In summary, in young pigs, adiponectin mRNA was up-regulated by fasting in visceral, but not s.c., adipose tissue, whereas AdipoR1 and AdipoR2 mRNA were increased in s.c., but not visceral, adipose tissue. The adiponectin receptor, T-cadherin, was expressed in s.c. and visceral adipose tissue and in muscle, but only muscle mRNA expression was decreased by fasting.