Preadipocyte Development Research Articles

Regulatory Effects of 198-bp Structural Variants in the GSTA2 Promoter Region on Adipogenesis in Chickens.

Molecular breeding accelerates animal breeding and improves efficiency by utilizing genetic mutations. Structural variations (SVs), a significant source of genetic mutations, have a greater impact on phenotypic variation than SNPs. Understanding SV functional mechanisms and obtaining precise information are crucial for molecular breeding. In this study, association analysis revealed significant correlations between 198-bp SVs in the GSTA2 promoter region and abdominal fat weight, intramuscular fat content, and subcutaneous fat thickness in chickens. High expression of GSTA2 in adipose tissue was positively correlated with the abdominal fat percentage, and different genotypes of GSTA2 exhibited varied expression patterns in the liver. The 198-bp SVs regulate GSTA2 expression by binding to different transcription factors. Overexpression of GSTA2 promoted preadipocyte proliferation and differentiation, while interference had the opposite effect. Mechanistically, the 198-bp fragment contains binding sites for transcription factors such as C/EBPα that regulate GSTA2 expression and fat synthesis. These SVs are significantly associated with chicken fat traits, positively influencing preadipocyte development by regulating cell proliferation and differentiation. Our work provides compelling evidence for the use of 198-bp SVs in the GSTA2 promoter region as molecular markers for poultry breeding and offers new insights into the pivotal role of the GSTA2 gene in fat generation.

COMBINATION OF ELECTROACUPUNCTURE THERAPY WITH HERBAL ROSEMARY (ROSEMARINUS OFFICINALIS L.) IN CASE OF OBESITY

Obesity or overweight is a condition that occurs when the quantity of body fat tissue compared to total body weight is greater than the normal state. Diet, exercise, psychological factors, heredity, hormones, and drug side effects are a few of the factors that might cause obesity. Gastric heat syndrome was present in the patient in this case study. The patients received electrostimulator-assisted acupuncture treatment 12 times for a total of 15 minutes at the following points: Fenglong (ST40), Neiting (ST44), Sanyinjiao (SP6), and Quchi (LI11), and was also given the herbal remedy rosemary (Rosmarinus officinalis L.) for 28 days, taken twice daily. Through the activation of PPAR- g, inhibition of prankeas lipase, and inhibition of pre-adipocyte development into adipocytes, carnosic acid, a component of rosemary, helps people lose weight. Additionally, carnosic acid stimulates the excretion of fat in feces. According to the case study results, the patient's body weight dropped from 77.6 kg to 73.9 kg. This indicates that acupuncture treatments at the points Fenglong (ST40), Neiting (ST44), Sanyinjiao (SP6), and Quchi (LI11) together with the administration of rosemary herbs can result in weight loss of up to 3.7 kg over the course of 28 days.

Sox6 impairs the adipogenic commitment of mesenchymal stem cells by targeting lysyl oxidase and preadipocyte factor 1

The commitment of mesenchymal stem cells (MSCs) to preadipocytes and the termination of differentiation to adipocytes are critical for maintaining systemic energy homeostasis. However, our knowledge of the molecular mechanisms governing the commitment of MSCs to preadipocytes and the subsequent termination of their differentiation into adipocytes remain limited. Additionally, the role of Sox6 sex-determining region Y (SRY)-box6 (Sox6), a transcription factor that regulates gene transcription, is reportedly involved in various cellular processes, including adipogenesis; however, its function in regulating preadipocyte development and the factors involved in the termination of adipogenic differentiation remain unexplored. Therefore, we investigated the role of Sox6 in regulating the differentiation of adipocytes by monitoring the effects of its overexpression in C3H10T1/2 cells (in vitro) and C57BL/6J mouse (in vivo) models of adipogenesis. We observed lower Sox6 expression in the adipose tissue of obese mice than that in control mice. Sox6 overexpression inhibited the differentiation of MSC by directly binding to the lysyl oxidase (Lox) and preadipocyte factor 1 (Pref1) promoters, which was potentiated by histone deacetylase-1(HDAC1). Our findings suggest that Sox6 is a key regulator of MSC commitment to adipocytes; therefore, targeting the Sox6-mediated regulation of this process could offer potential therapeutic avenues for addressing obesity and related metabolic disorders.

Vitamin A supplementation downregulates ADH1C and ALDH1A1 mRNA expression in weaned beef calves.

Our previous studies demonstrated that oral vitamin A supplementation during late-stage pregnancy and the neonatal stage enhances birth weight, growth performance, and mRNA expression related to muscle and preadipocyte development in beef cattle. The alcohol dehydrogenase 1C (ADH1C) c.-64T > C genotype also correlated with vitamin A concentration in beef production. This study aimed to investigate the effects of vitamin A supplementation on the muscle development and vitamin A metabolism in weaned beef calves with different ADH1C genotypes. Twenty male calves (90 d of age; initial BW: 89.03 kg [SD 8.60]) were stratified according to ADH1C genotype and vitamin A treatment (duration: 3 months) and randomly assigned to 4 groups with a 2 × 2 factorial arrangement. Vitamin A treatments included the following: control (10,000 IU/kg of as-fed, a. TT type; b. TC type); treatment (40,000 IU/kg of as-fed, c. TT type; and d. TC type). Parameters including BW, FI, blood, longissimus dorsi muscle, and liver status during the experimental period were analyzed using the generalized linear model (GLM) procedure and Tukey's test by SAS 9.4 program. Serum vitamin A was significantly increased (P < 0.05) in the vitamin A treatment group at 4 and 6 months of age. TT type calves showed higher serum vitamin A concentration (P < 0.05) than the TC type calves. Serum triglyceride and non-esterified fatty acid (NEFA) levels increased (P < 0.05) in the treatment group compared with the control at 6 months of age. However, BW, ADG and FI showed no differences between the groups. In addition, mRNA expression in longissimus dorsi muscle revealed upregulation of paired box 7 (PAX7) (P < 0.05) after the vitamin A treatment period based on biopsy results. Both ADH1C and aldehyde dehydrogenase (ALDH) 1A1 mRNA expression was downregulated (P < 0.01) by vitamin A supplementation. The TC type of ADH1C showed higher mRNA expression than the TT type. However, no effect was observed on adipogenic mRNA expression (preadipocyte factor-1 [PREF-1], peroxisome proliferator-activated receptor gamma [PPARγ], fatty acid binding protein 4 [FABP4]) in all groups. Our findings suggest that weaned calves treated with vitamin A may promote the storage of satellite cells by elevating PAX7 gene expression in the muscle. The TC type calves may show increased capacity for vitamin A metabolism, which can be used in genetically customizing feed management to maximize beef production in the calves.

An in vitro evaluation of luffa cylindrica stem sap in preadipocytes and dermal fibroblasts

BackgroundLuffa cylindrica stem sap (LuCS) has been ethnopharmacologically used as a cosmetic ingredients to improve the facial condition in Asians, but there is no scientific proof about the advantages of LuCS as a supplement for skin elasticity inducer. PurposePresently, we have validated the beneficial effect of LuCS in human preadipocyte and fibroblast. MethodsIn vitro activities of LuCS on expression of cellular elastin and collagen type I were validated using Western blot analysis in human fibroblasts. Effect of LuCS on preadipocyte development was performed using MDI medium containing isobutyl-methylxanthine, dexamethasone, and insulin and then evaluated using oil red O staining. ResultsTreatment of LuCS stimulated the expression of cellular elastin and type I procollagen in human skin fibroblasts. Exposure to LuCS induced lipid accumulation of preadipocytes via activation of CEBP/α signaling pathway in preadipocytes. Expression of collagen I, elastin, or CEBP/α mRNA was decreased by age. 3-bromo-3-methylisoxazol-5-amine enhanced the synthesis of cellular lipid in preadipocytes. ConclusionsCollectively, these results suggest the rationale of LuCS treatment in enhancing the skin condition.

Leptin promotes adipocytes survival in non-vascularized fat grafting via perfusion increase

BackgroundThough autologous fat transplantation is regularly and successfully performed in plastic surgery, little is known about the factors that contribute to the rise of preadipocytes and how the viability of adipocytes is regulated. As sufficient blood supply is a key parameter for the transplant's survival, we opted to analyse the development of preadipocytes within the fat transplant via stimulation of tissue perfusion with the angiogenesis enhancing hormone leptin. MethodsIn a murine (C57BL/6N) model inguinal fat was autologously transplanted into a dorsal skinfold chamber. In the intervention group the fat transplant was treated with local administration of leptin (3 μg/ml) at days 3, 7 and 10 after transplantation. Saline solution was administered respectively in the control group. On the postoperative days 3, 7, 10, and 15 intra vital microscopy was done to assess the functional vessel density, vessel diameter, adipocyte survival and preadipocyte development. The study was completed by histological tissue analysis on days 15 after transplantation. ResultsLeptin administration leads to an increase of angiogenesis, which starts from day 7 after implantation and elevates perfusion as well as functional vessel density FVD at days 10 and 15 after transplantation. Perfusion develops first from the border zones of the transplant. Histological evaluation showed that the percentage of perilipin positive adipocytes increased markedly in the study group of mice. Moreover, fat transplants of mice of the leptin group disclosed significantly higher Pref-1 positive cells than fat transplants of the control group. The findings reported in this study indicate that the leptin can enhance the survival and the quality of grafted fat tissue, which may be due to induction of angiogenesis. ConclusionLeptin administration to fat transplants induced an increase in angiogenesis in the transplanted tissue and may play a role in reducing the resorption rate of lipoaspirates.

Oral vitamin A supplementation during neonatal stage enhances growth, pre-adipocyte and muscle development in Korean native calves

Recently, many studies reported the critical importance of marbling development during late pregnancy and neonatal stage by influencing the preadipocyte hyperplasia. Our previous study demonstrated that 100,000 IU/day oral vitamin A supplementation during late-stage pregnancy enhances birth weight and gene expression-related muscle and preadipocyte development in offspring. This study investigated the effects of the oral supplementation of vitamin A on growth, and preadipocyte and muscle development in Korean native calves at the postnatal neonatal stage. Twenty-seven newborn calves were then randomly divided into two groups encompassing similar number of bulls and heifers (body weight of 31.6 ± 3.98 kg; age: from five days of birth to two-months age; control: 9 bulls and 4 heifers; treatment: 10 bulls and 4 heifers). All calves were treated with (treatment group) or without (control group) oral vitamin A supplementation (20,000 IU vs. 45,000 IU vitamin A/day, retinyl acetate) for two months until weaning. Vitamin A supplementation resulted in higher BW on day 45 (p < 0.05), as well as an upward tendency toward day 60 (weaned) in the treatment group (p = 0.079). Serum vitamin A showed an increasing trend on day 45 and 60 (p = 0.098 and p = 0.060, respectively) with vitamin A supplementation. However, no significant difference was found in complete blood counts analysis in treatment group compared to the control group. The mRNA expression results in longissimus dorsi muscle revealed that vitamin A supplementation promoted the levels of mRNA expression markers (p < 0.05) related to preadipocyte development (Pref-1, Zfp423, CTNNB1, ERK1, ERK2) and muscle development (MyoD, Myf6, Myogenin). High vitamin A supplementation during the postnatal period in newborn calves (45,000 IU/day) enhanced calf growth performance. In addition, according to the mRNA expression in two-month-old calves, positive impacts were observed on preadipocyte and muscle development with vitamin A supplementation. Collectively, vitamin A supplementation to calves can be beneficial for future production properties.

The effects of vitamin A supplementation during late-stage pregnancy on longissimus dorsi muscle tissue development, birth traits, and growth performance in postnatal Korean native calves.

ObjectiveThis study investigated the effects of vitamin A (VA) supplementation during late-stage pregnancy on longissimus dorsi muscle tissue development, birth traits, and growth performance of postnatal Korean native calves.MethodsIn the preliminary experiment, twenty-six pregnant cattle (initial body weight [BW] = 319 kg (standard deviation [SD] = 30.1; 1st parity) were randomly assigned to the control and treatment groups. The treatment group received VA supplementation at 24,000 IU/d from gestational day 225 until delivery. In the main experiment, twelve pregnant cattle (initial BW = 317 kg [SD = 31.3]; 1st parity) were treated with VA supplementation at 24,000 IU/d (gestational days 150 to 225) and at 78,000 IU/d (gestational day 225 until delivery). Serum VA levels were analyzed in pregnant cattle, and the growth performance, gene expression, and serum VA levels were analyzed in the offspring.ResultsSerum VA levels in pregnant cattle decreased the late gestation in both experiments (p<0.001). In the main experiment, pregnant cattle at parturition and offspring at birth in the treatment group had higher serum VA levels than those in the control group (p<0.05). In the treatment groups, an increased birth weight was observed in the main experimental group (p = 0.022), and a tendency (p = 0.088) toward an increased birth weight was observed in the preliminary experimental group. However, no differences were observed in the feed intake, average daily gain, gain-to-feed ratio, or BW of 31-day-old calves. Gene expression was analyzed in longissimus dorsi muscles of 31-day-old calves. VA supplementation in pregnant cattle stimulated postnatal muscle development in offspring by elevating myogenic factor 5 (MYF5), MYF6, and myoblast determination levels (p<0.05). Moreover, preadipocyte-related marker genes such as extracellular signal-regulated kinase 2 and krüppel-like factor 2 were higher in the treatment group than in the control group (p<0.05).ConclusionVA supplementation (78,000 IU/d) in late-stage pregnant cattle maintained serum VA levels. In addition, 78,000 IU/d VA supplementation increased the birth weight and expression of genes related to muscle and preadipocyte development in offspring. Overall, 78,000 IU/d VA supplementation in pregnant cattle is beneficial to newborn calves.

Bone morphogenetic protein 2 is a depot-specific regulator of human adipogenesis

BackgroundBone morphogenetic proteins (BMPs) regulate adipogenesis but it is not clear whether they influence regional adipose tissue (AT) development in humans.ObjectiveTo characterise BMP2 expression, BMP2-SMAD1/5/8 signalling, and BMP2′s potential effect on proliferation and adipogenesis in human subcutaneous abdominal and gluteal AT and its constituent preadipocytes.MethodsBMP2 expression was measured in whole AT and immortalised preadipocytes via qPCR and Western blot; secreted/circulating BMP2 was measured by ELISA. The effect of BMP2 on preadipocyte proliferation was evaluated using a fluorescent assay. BMP2′s effect on adipogenesis in immortalised preadipocytes was determined via qPCR of adipogenic markers and cellular triacylglycerol (TAG) accumulation. BMP2-SMAD1/5/8 signalling was assessed in immortalised preadipocytes via Western blot and qPCR of ID1 expression.ResultsBMP2 was expressed and released by abdominal and gluteal AT and preadipocytes. Exogenous BMP2 dose dependently promoted adipogenesis in abdominal preadipocytes only; 50 ng/ml BMP2 increased PPARG2 expression (10-fold compared to vehicle, p < 0.001) and TAG accumulation (3-fold compared to vehicle; p < 0.001). BMP2 stimulated SMAD1/5/8 phosphorylation and ID1 expression in abdominal and gluteal preadipocytes but this was blocked by 500 nM K02288, a type 1 BMP receptor inhibitor (p < 0.001). Co-administration of 500 nM K02288 also inhibited the pro-adipogenic effect of 50 ng/ml BMP2 in abdominal cells; >90% inhibition of TAG accumulation (p < 0.001) and ~50% inhibition of PPARG2 expression (p < 0.001). The endogenous iron regulator erythroferrone reduced BMP2-SMAD1/5/8 signalling by ~30% specifically in subcutaneous abdominal preadipocytes (p < 0.01), suggesting it plays a role in restricting the expansion of the body’s largest AT depot during energy deficiency. Additionally, a waist-hip ratio-increasing common polymorphism near BMP2 is an eQTL associated with ~15% lower BMP2 expression in abdominal and gluteal AT (p < 0.05) as well as altered adipocyte size in male abdominal AT (p < 0.05).ConclusionsThese data implicate BMP2-SMAD1/5/8 signalling in depot-specific preadipocyte development and abdominal AT expansion in humans.

MiR-425-5p Inhibits Differentiation and Proliferation in Porcine Intramuscular Preadipocytes

Intramuscular fat (IMF) content affects the tenderness, juiciness, and flavor of pork. An increasing number of studies are focusing on the functions of microRNAs (miRs) during porcine intramuscular preadipocyte development. Previous studies have proved that miR-425-5p was enriched in porcine skeletal muscles and played important roles in multiple physiological processes; however, its functions during intramuscular adipogenesis remain unclear. To explore the role of miR-425-5p in porcine intramuscular adipogenesis, miR-425-5p agomir and inhibitor were used to perform miR-425-5p overexpression and knockdown in intramuscular preadipocytes, respectively. Our results showed that the agomir of miR-425-5p dramatically inhibited intramuscular adipogenic differentiation and downregulated the expression levels of adipogenic marker genes PPARγ, FABP4, and FASN, whereas its inhibitor promoted adipogenesis. Interestingly, the agomir repressed proliferation of porcine intramuscular preadipocytes by downregulation of cyclin B and cyclin E. Furthermore, we demonstrated that miR-425-5p inhibited adipogenesis via targeting and repressing the translation of KLF13. Taken together, our findings identified that miR-425-5p is a novel inhibitor of porcine intramuscular adipogenesis possibly through targeting KLF13 and subsequently downregulating PPARγ.

Obesity-Driven Gut Microbiota Inflammatory Pathways to Metabolic Syndrome.

The intimate interplay between immune system, metabolism, and gut microbiota plays an important role in controlling metabolic homeostasis and possible obesity development. Obesity involves impairment of immune response affecting both innate and adaptive immunity. The main factors involved in the relationship of obesity with inflammation have not been completely elucidated. On the other hand, gut microbiota, via innate immune receptors, has emerged as one of the key factors regulating events triggering acute inflammation associated with obesity and metabolic syndrome. Inflammatory disorders lead to several signaling transduction pathways activation, inflammatory cytokine, chemokine production and cell migration, which in turn cause metabolic dysfunction. Inflamed adipose tissue, with increased macrophages infiltration, is associated with impaired preadipocyte development and differentiation to mature adipose cells, leading to ectopic lipid accumulation and insulin resistance. This review focuses on the relationship between obesity and inflammation, which is essential to understand the pathological mechanisms governing metabolic syndrome.

Expression profiling of preadipocyte microRNAs by deep sequencing on chicken lines divergently selected for abdominal fatness.

Through posttranscriptional gene regulation, microRNA (miRNA) is linked to a wide variety of biological processes, including adipogenesis and lipid metabolism. Although miRNAs in mammalian adipogenesis have been worked on extensively, their study in chicken adipogenesis is still very limited. To find miRNAs potentially important for chicken preadipocyte development, we compared the preadipocyte miRNA expression profiles in two broiler lines divergently selected for abdominal fat content, by sequencing two small RNA libraries constructed for primary preadipocytes isolated from abdominal adipose tissues. After bioinformatics analyses, from chicken miRNAs deposited in miRBase 20.0, we identified 225 miRNAs to be expressed in preadipocytes, 185 in the lean line and 200 in the fat line (derived from 208 and 203 miRNA precursors, respectively), which corresponds to 114 miRNA families. The let-7 family miRNAs were the most abundant. Furthermore, we validated the sequencing results of 15 known miRNAs by qRT-PCR, and confirmed that the expression levels of most miRNAs correlated well with those of Solexa sequencing. A total of 33 miRNAs was significantly differentially expressed between the two chicken lines (P<0.05). Gene ontology analysis revealed that they could target genes enriched in the regulation of gene transcription and chromatin function, response to insulin stimulation, and IGF-1 signaling pathways, which could have important roles in preadipocyte development. Therefore, a valuable information and resource of miRNAs on chicken adipogenesis were provided in this study. Future functional investigations on these miRNAs could help explore related genes and molecular networks fundamental to preadipocyte development.

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: Extracellular matrix in skeletal muscle development and meat quality1

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: Extracellular matrix in skeletal muscle development and meat quality1

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: The influence of extracellular matrix on intramuscular and extramuscular adipogenesis1

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.

Cytokines Promote Wnt Signaling and Inflammation and Impair the Normal Differentiation and Lipid Accumulation in 3T3-L1 Preadipocytes

Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFalpha impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein alpha, in contrast to peroxisome proliferator-activated receptor gamma, was markedly reduced. TNFalpha also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFalpha maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and beta-catenin levels. TNFalpha, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFalpha prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation.

Coculture of endothelial cells and mature adipocytes actively promotes immature preadipocyte development in vitro.

Adipose tissue consists of mature adipocytes and endothelial cells, which are all supported by the extracellular matrix. Adipose tissue development is closely associated with angiogenesis. However, the adipocyte-endothelial cell interaction is unclear. To address this issue, we examined the effects of endothelial cells on the growth, apoptosis, and differentiation of mature adipocytes in three-dimensional collagen gel culture of the adipocytes with or without rat lung endothelial (RLE) cells. Spindle-shaped preadipocytes, an immature type of adipocyte, developed more actively around the adhesion sites of RLE cells to mature adipocytes in the coculture (rate of preadipocytes: 18.9+/-4.3%) than in the culture of adipocytes alone (2.0+/-5.1%). With respect to growth, RLE cells induced about a three-fold increase in bromodeoxyuridine uptake of mature adipocytes alone, while RLE cells did not influence the uptake of preadipocytes. RLE cells also did not affect the apoptotic indices by immunohistochemistry for single-stranded DNA in mature adipocytes or preadipocytes. These phenomena were not reproduced by RLE cell-conditioned medium, or by certain endothelial cell-produced cytokines. Our in vitro study is the first demonstration that endothelial RLE cells promote the active development of preadipocytes together with increased growth of mature adipocytes. These results suggest that endothelial cells are involved in the enlargement mechanism of adipose tissue mass through their direct adhesion to mature adipocytes.

Tumor necrosis factor-alpha stimulates cell proliferation in adipose tissue-derived stromal-vascular cell culture: promotion of adipose tissue expansion by paracrine growth factors.

Elevated levels of tumor necrosis factor-alpha (TNF-alpha) protein and mRNA have been reported in adipose tissue from obese humans and rodents. However, TNF-alpha has catabolic and antiadipogenic effects on adipocytes. Addressing this paradox, we tested the hypothesis that paracrine levels of TNF-alpha, alone or together with insulin-like growth factor-I (IGF-I), support preadipocyte development. Cultured stromal-vascular cells from rat inguinal fat depots were exposed to serum-free media containing insulin and 0.2 nM TNF-alpha, 2.0 nM TNF-alpha, or 0.2 nM TNF-alpha + 1.0 nM IGF-I at different times during 7 days of culture. TNF-alpha 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-alpha did not have a long term effect on inhibiting differentiation. Continuous exposure to 0.2 nM TNF-alpha 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-alpha + 1 nM IGF-I on increasing cell number by Day 7 of culture to levels greater than those observed with either treatment applied alone. These data suggest that paracrine levels (0.2 nM) of TNF-alpha 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.

Adipocyte development is dependent upon stem cell recruitment and proliferation of preadipocytes.

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. Freshly isolated S-V cells from rat inguinal adipose tissues were plated for 24 hours then exposed to serum-free medium. 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'-deoxy-uridine 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. 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.

Influence of thyroxine in vivo on preadipocyte development and insulin-like growth factor-I and IGF binding protein secretion in fetal stromal vascular cell cultures.

Studies were conducted to determine the influence of thyroxine (T4) in vivo on preadipocyte development and insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) secretion in stromal-vascular (S-V) cultures. Fetal pigs were hypophysectomized (hypox) at 70 days of gestation, implanted with T4 pellets, and fetuses from the dam at 75 days of gestation. In a second experiment, hypox and T4 implantation were performed at 75 days and fetal pigs removed at 95 days of gestation. Primary cultures of stromal vascular (S-V) cells derived from fetal adipose tissue were established. Cultures were stained for morphological analysis and conditioned media were collected for IGF-1 determination by radioimmunoassay (RIA) and IGFBP analysis by Western blotting. After only 5 days of T4 treatment, fat cell cluster number and size and lipid deposition in cultures were significantly increased compared to cultures from untreated hypox fetuses. Fetal hypox reduced IGF-I secretion by preadipocytes at both ages and T4 treatment completely normalized IGF-I secretion (p < 0.05). Four IGFBPs (BP-1, BP-2, BP-3 & BP-4) detected in S-V cultures derived from 95-day fetuses were decreased in concentration by hypox by 44 +/- 9.4%, 32 +/- 9.7%, 42 +/- 12% and 53 +/- 6.9%. In cultures derived from T4 treated hypox fetuses, the levels of these four IGFBPs were increased by 187 +/- 25%, 239 +/- 38%, 190 +/- 5% and 347 +/- 43% over control values, respectively. In cultures from 75-day fetuses, only IGFBP-2 (major one) and BP-1 (minor one) were detected and their secretion was also decreased by hypox and elevated by T4 treatment (190 +/- 49.5%, 156 +/- 30%, respectively, of controls). The results provide direct evidence that T4 has a major influence on fetal preadipocyte development. T4 stimulated production of IGF-I and IGFBP in fetal S-V cultures, which in turn, may have mediated the capability of T4 to enhance fetal adipose tissue development.

The influence of extracellular matrix substrata on preadipocyte development in serum-free cultures of stromal-vascular cells.

The influence of the extracellular matrix (ECM) and ECM components on preadipocyte development was examined in primary cultures of adipose tissue stromal-vascular (S-V) cells. Extracellular matrix derived from Engelbreth-Holm-Swarm (EHS) cells or tumors enhanced several aspects of adipogenesis in vitro. In comparison to uncoated and fibronectin substrata, EHS-ECM substratum markedly increased attachment, spreading, and hypertrophy of preadipocytes while antagonizing spreading of non-preadipocytes. In addition, adipocyte number increased (P < .05) on these substrata despite no increase in total cell number: this resulted in a greater (P < .05) proportion of preadipocytes. These effects of EHS-ECM were also observed with laminin substrata per se, whereas types I and IV collagen and fibronectin had no influence. In contrast to all other substrata, adipocyte number decreased and total cell number increased 2.5-fold on ECM derived from corneal endothelial cells; this resulted in the lowest proportion of preadipocytes. Challenging cultures with adipogenic media (+serum) did not counter the inhibitory influence of corneal endothelial ECM, whereas dexamethasone partially neutralized the inhibitory influence of this ECM. These studies clearly show that source or type of the ECM dictated the influence of ECM substrata on preadipocyte development in primary S-V cultures. However, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development.