Adipose Conversion Research Articles

Positive effect of collagen V and VI on triglyceride accumulation during differentiation in cultures of bovine intramuscular adipocytes

Ethyl-3,4-dihydroxybenzoate (EDHB), a specific inhibitor of collagen synthesis, was used to study the role of collagen in the differentiation of bovine intramuscular preadipocytes (BIP). Triglyceride (TG) accumulation levels of BIP cells were dose-dependently inhibited by EDHB and were reduced to 50 % at a 0.1 mM concentration. EDHB addition prevented the accretion of collagens (types I–VI) on the cell surface, which generally increases during adipose conversion. Western blotting and immunofluorescence studies showed in detail that triple-helical conformation of procollagen molecules was drastically interrupted by EDHB, and as a result, their matrix assembly was not performed in the extracellular space of adipocytes. Particularly, the development of collagen types IV, V and VI during differentiation was severely damaged. When exogenous collagens were supplied to make up for the lack of endogenous products, cultured EDHB-treated cells on type V and VI collagen-coated dishes were the only ones among six collagens to accumulate more TG, although their TG content did not reach that of normal adipocytes. This result implies the importance and the active role of collagens V and VI for adipogenesis. However, these findings also indicate that collagen newly synthesized and organized by the adipocyte itself during differentiation is still necessary for the growth of adipose tissue.

Identification of novel putative membrane proteins selectively expressed during adipose conversion of 3T3-L1 cells

Fat tissue plays a critical role in the regulation of energy metabolism. Here we report the proteomic identification of a novel fa t tissue-specific l ow molecular weight p rotein (Falp) which responds to insulin. Falp is preferentially expressed in adipocytes but not in preadipocytes, as shown by two-dimensional gel electrophoresis. Northern blot analysis shows that the Falp gene is predominantly expressed in brown and white fat tissues, but not in any other tissues examined. Human homologs of mouse Falp are found to exist as two alternatively spliced isoforms, which share the same N-terminus but have different C-termini. Both human and mouse Falp contain a conserved putative transmembrane domain. Immunofluorescent analyses of 3T3-L1 adipocytes show that Falp protein strictly localizes at a compact perinuclear membrane compartment. Treatment of cells with insulin induces the redistribution of Falp into numerous discrete spotty structures spreading throughout the cytoplasm. Whereas the function of Falp is currently unclear, its tissue specific expression and the responsiveness to insulin suggest that Falp might be involved in a process specifically restricted to adipose tissue function, such as vesicular transport and protein secretion.

Extracellular matrix development during differentiation into adipocytes with a unique increase in type V and VI collagen

In order to study how adipose conversion affects the extracellular environment, levels of extracellular matrix (ECM) proteins during differentiation were analyzed by 125I-labeled antibody binding to each specific primary antibody. When confluent bovine intramuscular preadipocytes (BIP) were stimulated with adipogenic medium, there was a significant accretion on the cell surface of type I–VI collagens, laminin and fibronectin, compared with undifferentiated cells. The deposition amount of ECM proteins had reached near maximal levels at an early stage of differentiation and lasted throughout the culture. However, the increasing manners were not all the same in these eight proteins. Type V and type VI collagen tended to show a transient decline after the rapid rise at the beginning of stimulation, and fibronectin instead, subsequently decreased. Further analysis by immunocytochemical staining showed that remodeling occurred in type V and VI collagen matrices during this period; extensive fibrillar networks seen at 10 d after stimulation were quite unlike that formed earlier. These specific increases and development of matrix during adipocyte differentiation imply some significance for organizing fat lobules in each ECM proteins, especially type V and VI collagens.

Proprotein convertases are important mediators of the adipocyte differentiation of mouse 3T3-L1 cells.

Mouse 3T3-L1 cells are widely used to study adipocyte differentiation in vitro. When treated with insulin, dexamethasone and isobutylmethylxanthine these fibroblastic cells differentiate into round triglyceride-rich adipocytes. Because several proteins implicated in adipocyte differentiation (e.g. type 1 IGF receptors) are proteolytically activated by endoproteinases of the proprotein convertase family, we sought to determine whether these endoproteinases are crucial for adipose conversion. In this study, we show that expression of the proprotein convertases PACE4, PC7 and furin increases when 3T3-L1 cells are induced to differentiate into adipocytes. The differentiation was blocked in transfected cells expressing alpha1-antitrypsin Portland or in normal cells pre-treated with the synthetic inhibitor decanoyl-RVKR-chloromethylketone. Both inhibitors are known to specifically inactivate proprotein convertases. The block was associated with impaired proteolytic activation of proIGF-1 receptor, absence of induction of the adipogenic transcriptional factor PPARgamma and marked reduction of the nuclear translocation of the C/EBPbeta factor. Taken together, these data constitute evidence that proprotein convertases are crucial mediators of adipogenesis.

Vitamin C is one of the lipolytic substances in green tea

We have studied the influence of vitamin C contained in green tea on the lipolysis of well-differentiated 3T3-L1 cells. When mature adipocytes were exposed to vitamin C the triglyceride concentration was decreased (p < 0.05) and the activity of glycerophosphate dehydrogenase, a marker of adipose conversion, was significantly inhibited (p < 0.01). These data suggest that green tea may have a lipolytic activity due to the mechanism by which the vitamin C contained in it inhibits triglyceride accumulation.

Dehydrotrametenolic acid induces preadipocyte differentiation and sensitizes animal models of noninsulin-dependent diabetes mellitus to insulin.

We recently discovered that the triterpene acid compound dehydrotrametenolic acid promotes adipocyte differentiation in vitro and acts as an insulin sensitizer in vivo. This natural product has been isolated from dried sclerotia of Poria cocos WOLF (Polyporaceae), a well-known traditional Chinese medicinal plant. We examined the effects of dehydrotrametenolic acid on plasma glucose concentration in obese hyperglycemic db/db mice. Dehydrotrametenolic acid can reduce hyperglycemia in mouse models of noninsulin-dependent diabetes mellitus (NIDDM) and act as an insulin sensitizer as indicated by the results of the glucose tolerance test. These terpenoids and thiazolidine type of antidiabetic agents such as Ciglitazone, although structurally unrelated, share many biological activities: both induce adipose conversion, activate peroxisome proliferator-activated receptor gamma (PPAR gamma) in vitro, and reduce hyperglycemia in animal models of NIDDM. Dehydrotrametenolic acid is a promising candidate for a new type of insulin-sensitizing drug. This finding is very important for the development of insulin sensitizers that are not of the thiazolidine type.

Molecular cloning, expression analysis, promoter characterization, and chromosomal localization of the bovine PREF1 gene.

Pre-adipocyte factor-1 (pref-1), originally identified in mouse 3T3-L1 pre-adipocytes, is known to play a key role in inhibiting the adipose conversion. As a first step to study the involvement of the PREF1 gene in intramuscular adipose tissue development in cattle, which has an economic importance for beef cattle, we have isolated the bovine PREF1 cDNA and genomic clones and characterized expression in abdominal adipose and promoter region of the bovine PREF1 gene. We have detected two bovine PREF1 splice-isoforms, PREF1C2 and PREF1A, in abdominal fat tissue. The RT-PCR experiment revealed that the two isoforms are identified in neonatal, but no in adult abdominal fat tissue, suggesting age-dependent suppression of the bovine PREF1 gene expression in the form of PREF1C2 and PREF1A in abdominal fat tissue. By mapping the regulatory region of this gene, we have shown that at least two regions within 1121 bp upstream of putative transcription start site are sufficient to confer promoter activity, when accompanied by a short region including the transcription start site. The chromosomal location of the PREF1 gene was determined by fluorescence in situ hybridization (FISH). The PREF1 gene locates on bovine chromosome 21q24.

Tumor Necrosis Factor-α-induced Adipose-related Protein (TIARP), a Cell-surface Protein That Is Highly Induced by Tumor Necrosis Factor-α and Adipose Conversion

Tumor necrosis factor-alpha (TNF alpha) is involved in the physiological and biological abnormalities found in two opposite metabolic situations: cachexia and obesity. In an attempt to identify novel genes and proteins that could mediate the effects of TNFalpha on adipocyte metabolism and development, we have used a differential display technique comparing 3T3-L1 cells exposed or not to the cytokine. We have isolated a novel adipose cDNA encoding a TNF alpha-inducible 470-amino acid protein termed TIARP, with six putative transmembrane regions flanked by a large amino-terminal and a short carboxyl-terminal domain, a structure reminiscent of channel and transporter proteins. Commitment into the differentiation process is required for cytokine responsiveness. The differentiation process per se is accompanied by a sharp emergence of TIARP mRNA transcripts, in parallel with the expression of the protein at the plasma membrane. Transcripts are present at high levels in white and brown adipose tissues, and are also detectable in liver, kidney, heart, and skeletal muscle. Whereas the biological function of TIARP is presently unknown, its pattern of expression during adipose conversion and in response to TNF alpha exposure as a transmembrane protein mainly located at the cell surface suggest that TIARP might participate in adipocyte development and metabolism and mediate some TNF alpha effects on the fat cell as a channel or a transporter.

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of α1(IV) and α2(IV) mRNAs. Prolyl 4-hydroxylase α increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase α 2β 2 tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.

Adiponutrin, a Transmembrane Protein Corresponding to a Novel Dietary- and Obesity-linked mRNA Specifically Expressed in the Adipose Lineage

We have used a mRNA differential display technique to identify new genes involved in the reprogramming of gene expression during the adipose conversion of mouse 3T3 preadipocyte cell lines. We report here on the identification and cloning of a novel adipose-specific cDNA encoding a predicted membrane protein of 413 amino acids. The level of the corresponding 3.2-kilobase mRNA is tremendously increased during 3T3-L1 and 3T3-F442A differentiation into adipocytes. A single, very abundant 3.2-kilobase transcript is also found in inguinal and epididymal white adipose tissues and in interscapular brown adipose tissue but not in any other tissues examined. Its expression in adipose tissue is under tight nutritional regulation. The level of this novel 3.2-kilobase transcript becomes virtually nondetectable during fasting but is dramatically increased when fasted mice are refed a high carbohydrate diet. Based on its adipose specificity and dietary regulation, the novel gene product has been designated adiponutrin. The expression of adiponutrin mRNA is also 50-fold elevated in genetically obese fa/fa rats, indicating a link between adiponutrin and obesity. Western blot and confocal imagery analyses with epitope-tagged protein transiently expressed in 3T3-L1 adipocytes, and COS cells show that adiponutrin strictly localizes to membranes and is absent from the cytosol. Sequence analysis reveals homologies with several other members of related eukaryotic proteins, including a human paralog, which has been recently described in vesicular transport mechanisms. This leads us to suggest that adiponutrin could be involved in vesicular targeting and protein transport restricted to the adipocyte function.

Amine oxidase substrates mimic several of the insulin effects on adipocyte differentiation in 3T3 F442A cells.

We have previously reported that substrates of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) exert short-term insulin-like effects in rat adipocytes, such as stimulation of glucose transport. In the present work, we studied whether these substrates could also mimic long-term actions of insulin. Adipose differentiation of 3T3 F442A cells, which is highly insulin-dependent, served as a model to test the effects of sustained administration of amine oxidase substrates. Daily treatment of confluent cells with 0.75 mM tyramine (a substrate of MAO and SSAO) or benzylamine (a substrate of SSAO) over 1 week caused the acquisition of typical adipocyte morphology. The stimulation of protein synthesis and triacylglycerol accumulation caused by tyramine or benzylamine reached one half of that promoted by insulin. This effect was insensitive to pargyline (an MAO inhibitor), but was inhibited by semicarbazide (an SSAO inhibitor) and by N-acetylcysteine (an antioxidant agent), suggesting the involvement of the H(2)O(2) generated during SSAO-dependent amine oxidation. Chronic administration of amine oxidase substrates also induced the emergence of adipose conversion markers, such as aP2, glycerol-3-phosphate dehydrogenase, the glucose transporter GLUT4, and SSAO itself. Moreover, cells treated with amines acquired the same insulin sensitivity regarding glucose transport as adipocytes classically differentiated with insulin. In all, most of the adipogenic effects of amines were additive to insulin. Our data reveal that amine oxidase substrates partially mimic the adipogenic effect of insulin in cultured preadipocytes. Furthermore, they suggest that SSAO not only represents a novel late marker of adipogenesis, but could also be directly involved in the triggering of terminal adipocyte differentiation.

Amine oxidase substrates mimic several of the insulin effects on adipocyte differentiation in 3T3 F442A cells

We have previously reported that substrates of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) exert short-term insulin-like effects in rat adipocytes, such as stimulation of glucose transport. In the present work, we studied whether these substrates could also mimic long-term actions of insulin. Adipose differentiation of 3T3 F442A cells, which is highly insulin-dependent, served as a model to test the effects of sustained administration of amine oxidase substrates. Daily treatment of confluent cells with 0.75mM tyramine (a substrate of MAO and SSAO) or benzylamine (a substrate of SSAO) over 1 week caused the acquisition of typical adipocyte morphology. The stimulation of protein synthesis and triacylglycerol accumulation caused by tyramine or benzylamine reached one half of that promoted by insulin. This effect was insensitive to pargyline (an MAO inhibitor), but was inhibited by semicarbazide (an SSAO inhibitor) and by N-acetylcysteine (an antioxidant agent), suggesting the involvement of the H2O2 generated during SSAO-dependent amine oxidation. Chronic administration of amine oxidase substrates also induced the emergence of adipose conversion markers, such as aP2, glycerol-3-phosphate dehydrogenase, the glucose transporter GLUT4, and SSAO itself. Moreover, cells treated with amines acquired the same insulin sensitivity regarding glucose transport as adipocytes classically differentiated with insulin. In all, most of the adipogenic effects of amines were additive to insulin. Our data reveal that amine oxidase substrates partially mimic the adipogenic effect of insulin in cultured preadipocytes. Furthermore, they suggest that SSAO not only represents a novel late marker of adipogenesis, but could also be directly involved in the triggering of terminal adipocyte differentiation.

The HIV protease inhibitor indinavir impairs sterol regulatory element-binding protein-1 intranuclear localization, inhibits preadipocyte differentiation, and induces insulin resistance.

Protease inhibitors used in the treatment of HIV infection have been causally associated with lipodystrophy and insulin resistance and were shown to alter adipocyte differentiation in cultured cells. We aimed to delineate the mechanism by which indinavir impaired adipocyte function. We report that indinavir altered neither the growth nor insulin sensitivity of 3T3-F442A preadipocytes, nor did it alter the initial step of their differentiation, i.e., clonal proliferation. However, adipose conversion was inhibited by indinavir (by 50-60%), as shown by 1) the decrease in the number of newly formed adipocytes; 2) the lower level of the adipogenic protein markers, sterol regulatory element-binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and the insulin receptor (IR); and 3) the lack of SREBP-1 and PPAR-gamma immunoreactivity in the nucleus of most indinavir-treated cells. Partial adipose conversion also correlated with an accumulation of SREBP-1 at the nuclear periphery and an alteration in its electrophoretic mobility. Defective expression and nuclear localization of PPAR-gamma probably resulted from the decreased level of nuclear SREBP-1. Indinavir also rendered 3T3-F442A adipocytes resistant to insulin for mitogen-activated protein kinase activation at a step distal to IR substrate-1 tyrosine phosphorylation. Hence, indinavir impairs differentiation at an early step of adipose conversion probably involving the process controlling SREBP-1 intranuclear localization.

Effects of angiotensin II on adipose conversion and expression of genes of the renin-angiotensin system in human preadipocytes.

A complete functional renin-angiotensin system exists in human adipose tissue, but its regulation and the effects of angiotensin II on cells from this tissue are only beginning to be understood. In this study, we examined the effects of angiotensin II on changes in lipid accumulation, specific glycerol-3-phosphate dehydrogenase activity, and the expression of five genes of the renin-angiotensin system during the adipose conversion of human primary cultured preadipocytes. Angiotensin II leads to a distinct reduction in insulin-induced differentiation, but only has a marginal effect on the adipose conversion of cells stimulated with insulin, cortisol, and isobutyl methyl xanthine. During differentiation, angiotensinogen mRNA levels rise, renin mRNA levels decline, whereas renin-binding protein and angiotensin-converting enzyme levels are unaffected. Angiotensin II downregulates angiotensinogen and renin gene expression, but it does not affect renin-binding protein and angiotensin-converting enzyme levels. Angiotensin II thus prevents the development of adipocytes in contact with high insulin levels, while not inhibiting differentiation, which is further stimulated. Therefore, angiotensin II could be a protective factor against uncontrolled expansion of adipose tissue. Further studies are needed to find out whether the effects of angiotensin II on the renin-angiotensin system are direct feedback loops or secondary to changes in the differentiation program.

Garcinia extract inhibits lipid droplet accumulation without affecting adipose conversion in 3T3-L1 cells.

Garcinia extract was isolated from the fruit of the Garcinia cambogia and was used as a potential antiobesity agent. In week 3 of culture with insulin, the fat cells exhibited more numerous and larger intracytoplasmic lipid droplets (i.e. 30-40 microm(2)). When Garcinia extract and insulin were added simultaneously, the accumulation of lipid droplets was inhibited and the peak droplet area shifted to become smaller (10-20 microm(2)). The activities of glycerophosphate dehydrogenase, a marker of adipose differentiation, were not significantly inhibited by the Garcinia extract. These findings suggest that the Garcinia extract inhibits lipid droplet accumulation in fat cells without affecting adipose conversion.

A plasma kallikrein-dependent plasminogen cascade required for adipocyte differentiation.

Here we show that plasma kallikrein (PKal) mediates a plasminogen (Plg) cascade in adipocyte differentiation. Ecotin, an inhibitor of serine proteases, inhibits cell-shape change, adipocyte-specific gene expression, and lipid accumulation during adipogenesis in culture. Deficiency of Plg, but not of urokinase or tissue-type plasminogen activator, suppresses adipogenesis during differentiation of 3T3-L1 cells and mammary-gland involution. PKal, which is inhibited by ecotin, is required for adipose conversion, Plg activation and 3T3-L1 differentiation. Human plasma lacking PKal does not support differentiation of 3T3-L1 cells. PKal is therefore a physiological regulator that acts in the Plg cascade during adipogenesis. We propose that the Plg cascade fosters adipocyte differentiation by degradation of the fibronectin-rich preadipocyte stromal matrix.

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.

Site-specific control of rat preadipocyte adipose conversion by ovarian status: Possible involvement of CCAAT/enhancer-binding protein transcription factors.

The preadipocyte-adipocyte conversion process from two intraabdominal (parametrial and perirenal fat depots) is differently affected by ovarian status in the rat. We have tested the hypothesis that these site-specific alterations of adipogenesis might be related to changes in the expression of the transcription factors c-mycand CCAAT/enhancer binding proteins (C/EBPalpha, -beta, and -zeta) that regulate proliferation and differentiation. The increased proliferation rates observed in parametrial and perirenal preadipocytes after ovariectomy were not linked to variations in c-myc mRNA levels. Expression of the early marker of adipogenesis, lipoprotein lipase (LPL), remained insensitive to the ovarian status in early differentiated parametrial and perirenal preadipocytes. By contrast, LPL expression increased in early differentiated sc preadipocytes from ovariectomized rats, an effect that was completely reversed by in vivo estradiol and progesterone treatment. Expression of C/EBPbeta protein was unaffected by ovarian status whatever the anatomic origin of the preadipocytes. By contrast, the levels of p42 and p30 isoforms of C/EBPalpha were specifically decreased in parametrial preadipocytes, an alteration that was completely corrected by in vivo administration of estradiol and progesterone. C/EBPzeta, a dominant inhibitor of C/EBPalpha and -beta, exhibited a strong site-specific expression since C/ EBPzeta content was fivefold higher in sc preadipocytes than in deep intraabdominal cells whatever the ovarian status. Furthermore, ovariectomy selectively decreased C/EBPzeta levels in sc cells. In conclusion, our study suggests that some of the site-specific effects of ovariectomy on adipogenesis could involve, at least in part, altered expressions of C/EBPalpha and -zeta, both of which are important transcriptional regulators of fat cell differentiation and metabolism.

Function of pref-1 as an inhibitor of adipocyte differentiation.

During conversion of preadipocytes to adipocytes, growth arrest and subsequent activation of adipocyte genes by the transcription factors, C/EBPalpha and PPARgamma, lead to adipogenesis. During differentiation, these cells not only start expressing those genes necessary for adipocyte function, but also undergo changes in morphology to become rounded lipid filled adipocytes. Various factors in cell-cell communication or cell-matrix interaction may govern whether preadipocytes are kept in an undifferentiated state or undergo differentiation. In an attempt to identify molecules that play critical roles in the conversion of preadipocytes to adipocytes, we cloned by differential screening several regulatory molecules, including pref-1. Pref-1 is an inhibitor of adipocyte differentiation and is synthesized as a plasma membrane protein containing 6 EGF-repeats in the extracellular domain. Pref-1 is highly expressed in 3T3-L1 preadipocytes, but is not detectable in mature fat cells. Dexamethasone, a component of standard differentiation agents, inhibits pref-1 transcription and thereby promotes adipogenesis. Downregulation of pref-1 is required for adipose conversion and constitutive expression of pref-1 inhibits adipogenesis. Conversely, decreasing pref-1 levels by antisense pref-1 transfection greatly enhances adipogenesis. The ectodomain of pref-1 is cleaved to generate a biologically active 50kDa soluble form. There are four major forms of membrane pref-1 resulting from alternate splicing. Two of these forms which have a deletion that includes the putative processing site proximal to the membrane do not produce a biologically active soluble form. This indicates that alternate splicing may determine the range of action, juxtacrine or paracrine, of pref-1.

Inhibition of Lipogenesis by pycnogenol

The influence of pycnogenol on the adipose conversion of 3T3-L1 cells by insulin was studied. In week 3 of culture with insulin, pycnogenol was found to inhibit significantly the expression of glycerophosphate dehydrogenase (p < 0.01). This finding suggests that pycnogenol inhibits the accumulation of lipid droplets in adipose tissue. Copyright © 2000 John Wiley & Sons, Ltd.