Fat Cell Development Research Articles

Modulation of PGC-1 Coactivator Pathways in Brown Fat Differentiation through LRP130

The PGC-1 coactivators are important regulators of oxidative metabolism. We previously demonstrated that LRP130 is a binding partner of PGC-1alpha, required for hepatic gluconeogenesis. LRP130 is the gene mutated in Leigh syndrome French Canadian variant, a rare neurodegenerative disease. The importance of LRP130 in other, non-hepatocyte biology remains obscure. To better understand PGC-1 coactivator function in brown fat development, we explored the metabolic role of LRP130 in brown adipocyte differentiation. We show that LRP130 is preferentially enriched in brown fat compared with white, and induced in a PGC-1-dependent manner during differentiation. Despite intact PGC-1 coactivator expression, brown fat cells deficient for LRP130 exhibit attenuated expression of several genes characteristic of brown fat, including uncoupling protein 1. Oxygen consumption studies support a specific defect in proton leak due to attenuated uncoupling protein 1 expression. Notably, brown fat cell development common to both PGC-1 coactivators is governed by LRP130. Conversely, the cAMP response controlled by PGC-1alpha is not regulated by LRP130. These data implicate LRP130 in brown fat cell development and differentiation.

Macrophage PPARγ is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones

PPAR gamma is required for fat cell development and is the molecular target of antidiabetic thiazolidinediones (TZDs), which exert insulin-sensitizing effects in adipose tissue, skeletal muscle, and liver. Unexpectedly, we found that inactivation of PPAR gamma in macrophages results in the development of significant glucose intolerance plus skeletal muscle and hepatic insulin resistance in lean mice fed a normal diet. This phenotype was associated with increased expression of inflammatory markers and impaired insulin signaling in adipose tissue, muscle, and liver. PPAR gamma-deficient macrophages secreted elevated levels of factors that impair insulin responsiveness in muscle cells in a manner that was enhanced by exposure to FFAs. Consistent with this, the relative degree of insulin resistance became more severe in mice lacking macrophage PPAR gamma following high-fat feeding, and these mice were only partially responsive to TZD treatment. These findings reveal an essential role of PPAR gamma in macrophages for the maintenance of whole-body insulin action and in mediating the antidiabetic actions of TZDs.

Molecular processes during fat cell development revealed by gene expression profiling and functional annotation

In-depth bioinformatics analyses of expressed sequence tags found to be differentially expressed during differentiation of 3T3-L1 pre-adipocyte cells were combined with de novo functional annotation and mapping onto known pathways to generate a molecular atlas of fat-cell development.

Adipose tissue angiogenesis1,2

A review of adipose tissue angiogenesis includes the morphological and cytochemical development of adipose tissue vasculature and the concept of primitive fat organs. Spatial and temporal relationships between fetal vascular and fat cell development are discussed, including depot- and genetic-dependent arteriolar differentiation. The relationship between connective tissue deposition and elaboration of adipose tissue vasculature is discussed with respect to regulating adipocyte development in a depot-dependent manner. In vitro studies indicated that depot-dependent vascular traits may be attributable to intrinsic growth characteristics of adipose tissue endothelial cells. These studies indicate that adipogenesis may be regulated by factors that drive angiogenesis. Fundamental aspects of angiogenesis, including basement membrane breakdown, vasculogenesis, angiogenic remodeling, vessel stabilization, and vascular permeability were reviewed. Critical angiogenic factors include vascular endothelial growth factor (VEGF), VEGF receptors, angiopoietins (Ang), ephrins, matrix metalloproteinases, and the plasminogen enzymatic system. Vascular endothelial growth factor is the most critical factor because it initiates the formation of immature vessels and disruption of a single VEGF allele leads to embryonic lethality in mice. Expression of VEGF is influenced by hypoxia, insulin, growth factors, and several cytokines. Angiogenic factors secreted and/or produced by adipocytes or preadipocytes are discussed. Vascular endothelial growth factor expression and secretion by adipocytes is regulated by insulin and hypoxia, and is associated with adipose tissue accretion. Vascular endothelial growth factor accounts for most of the angiogenic activity of adipose tissue. The proposed role of leptin as an adipogenic factor is reviewed with respect to efficacy on various aspects of angiogenesis relative to other angiogenic factors. The VEGF and leptin genes are both hypoxia inducible, but potential links between VEGF and leptin gene expression have not been examined. Finally, several studies including a study of mice treated with antiangiogenic factors indicate that adipose tissue accretion can be controlled through the vasculature per se.

Adipose tissue angiogenesis.

A review of adipose tissue angiogenesis includes the morphological and cytochemical development of adipose tissue vasculature and the concept of primitive fat organs. Spatial and temporal relationships between fetal vascular and fat cell development are discussed, including depot- and genetic-dependent arteriolar differentiation. The relationship between connective tissue deposition and elaboration of adipose tissue vasculature is discussed with respect to regulating adipocyte development in a depot-dependent manner. In vitro studies indicated that depot-dependent vascular traits may be attributable to intrinsic growth characteristics of adipose tissue endothelial cells. These studies indicate that adipogenesis may be regulated by factors that drive angiogenesis. Fundamental aspects of angiogenesis, including basement membrane breakdown, vasculogenesis, angiogenic remodeling, vessel stabilization, and vascular permeability were reviewed. Critical angiogenic factors include vascular endothelial growth factor (VEGF), VEGF receptors, angiopoietins (Ang), ephrins, matrix metalloproteinases, and the plasminogen enzymatic system. Vascular endothelial growth factor is the most critical factor because it initiates the formation of immature vessels and disruption of a single VEGF allele leads to embryonic lethality in mice. Expression of VEGF is influenced by hypoxia, insulin, growth factors, and several cytokines. Angiogenic factors secreted and/or produced by adipocytes or preadipocytes are discussed. Vascular endothelial growth factor expression and secretion by adipocytes is regulated by insulin and hypoxia, and is associated with adipose tissue accretion. Vascular endothelial growth factor accounts for most of the angiogenic activity of adipose tissue. The proposed role of leptin as an adipogenic factor is reviewed with respect to efficacy on various aspects of angiogenesis relative to other angiogenic factors. The VEGF and leptin genes are both hypoxia inducible, but potential links between VEGF and leptin gene expression have not been examined. Finally, several studies including a study of mice treated with antiangiogenic factors indicate that adipose tissue accretion can be controlled through the vasculature per se.

The dawn of the SPPARMs?

Thiazolidinediones (TZDs) are used as antidiabetic agents in the treatment of type II diabetes. These compounds are ligands for the nuclear hormone receptor PPARgamma, which is highly expressed in adipose tissue. PPARgamma acts as a molecular switch in the process of fat cell development. The quest for the ideal antidiabetic agent is challenged by the need to develop PPARgamma ligands that improve insulin sensitivity, but do not promote fat cell formation. A newly described PPARgamma ligand may represent an initial step in this direction and could lead to improved agents for treating insulin resistance in type II diabetes.

C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway.

PPARgamma and C/EBPalpha are critical transcription factors in adipogenesis, but the precise role of these proteins has been difficult to ascertain because they positively regulate each other's expression. Questions remain about whether these factors operate independently in separate, parallel pathways of differentiation, or whether a single pathway exists. PPARgamma can promote adipogenesis in C/EBPalpha-deficient cells, but the converse has not been tested. We have created an immortalized line of fibroblasts lacking PPARgamma, which we use to show that C/EBPalpha has no ability to promote adipogenesis in the absence of PPARgamma. These results indicate that C/EBPalpha and PPARgamma participate in a single pathway of fat cell development with PPARgamma being the proximal effector of adipogenesis.

Serpent, a GATA-like transcription factor gene, induces fat-cell development in Drosophila melanogaster.

The GATA-like transcription factor gene serpent is necessary for embryonic fat-cell differentiation in Drosophila (Sam, S., Leise, W. and Hoshizaki, D. K. (1996) Mech. Dev. 60, 197-205) and has been proposed to function in a cell-fate choice between fat cell and somatic gonadal precursors (Moore, L. A., Broihier, H. T., Van Doren, M. and Lehmann, R. (1998) Development 125, 837-44; Riechmann, V., Irion, U., Wilson, R., Grosskortenhaus, R. and Leptin, M. (1997) Development 124, 2915-22). Here, we report that deregulated expression of serpent in the mesoderm induces the formation of ectopic fat cells and prevents the migration and coalescence of the somatic gonadal precursors. The ectopic fat cells do not arise from hyperproliferation of the primary fat-cell clusters but they do associate with the endogenous fat cells to form a fat body that is expanded in both the dorsal/ventral and anterior/posterior axes. Misexpression of serpent also affects the differentiation of muscle cells. Few body-wall muscle precursors are specified and there is a loss of most body-wall muscle fibers. The precursors of the visceral mesoderm are also absent and concomitantly the visceral muscle is absent. We suggest that the ectopic fat cells might originate from cells that have the potential, but do not normally, differentiate into fat cells or from cells that have acquired a fat-cell fate. In light of our results, we discuss the role of serpent in fat-cell specification and in cell fate choices.

Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation.

To develop and to characterize a human preadipocyte cell strain with high capacity for adipose differentiation serving as a model for studying human adipocyte development and metabolism in vitro. Cells were derived from the stromal cells fraction of subcutaneous adipose tissue of an infant with Simpson-Golabi-Behmel syndrome (SGBS). Adipose differentiation was induced under serum-free culture conditions by exposure to 10 nM insulin, 200 pM triiodothyronine, 1 microM cortisol and 2 microM BRL 49653, a PPAR gamma agonist. During the differentiation process SGBS cells developed a gene expression pattern similar to that found in differentiating human preadipocytes with a characteristic increase in fat cell-specific mRNAs encoding lipoprotein lipase (LPL), glycero-3-phosphate dehydrogenase (GPDH), GLUT4, leptin and others. Differentiated SGBS cells exhibited an increase in glucose uptake upon insulin stimulation and in glycerol release upon catecholamine exposure. SGBS adipocytes were morphologically, biochemically and functionally identical to in vitro differentiated adipocytes from healthy subjects. However, while preadipocytes from healthy control infants rapidly lost their capacity to differentiate after a few cell divisions in culture, SGBS cells maintained their differentiation capacity over many generations: upon appropriate stimulation 95% of SGBS cells of generation 30 developed into adipocytes. A mutation in the glypican 3 gene was not detected in the patient. Thus, it remains unclear whether the molecular alteration in SGBS cells is also responsible for the high differentiation capacity and further investigations are required. The human cell strain described here provides an almost unlimited source of human preadipocytes with high capacity for adipose differentiation and may, therefore, represent a unique tool for studying human fat cell development and metabolism. International Journal of Obesity (2001) 25, 8-15

Peroxisome proliferator-activated receptor gamma ligands inhibit development of atherosclerosis in LDL receptor-deficient mice.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates fat-cell development and glucose homeostasis and is the molecular target of a class of insulin-sensitizing agents used for the management of type 2 diabetes mellitus. PPARgamma is highly expressed in macrophage foam cells of atherosclerotic lesions and has been demonstrated in cultured macrophages to both positively and negatively regulate genes implicated in the development of atherosclerosis. We report here that the PPARgamma-specific agonists rosiglitazone and GW7845 strongly inhibited the development of atherosclerosis in LDL receptor-deficient male mice, despite increased expression of the CD36 scavenger receptor in the arterial wall. The antiatherogenic effect in male mice was correlated with improved insulin sensitivity and decreased tissue expression of TNF-alpha and gelatinase B, indicating both systemic and local actions of PPARgamma. These findings suggest that PPARgamma agonists may exert antiatherogenic effects in diabetic patients and provide impetus for efforts to develop PPARgamma ligands that separate proatherogenic activities from antidiabetic and antiatherogenic activities.

The phosphodiesterase inhibitor IBMX suppresses TNF-alpha expression in human adipocyte precursor cells: a possible explanation for its adipogenic effect.

Tumor necrosis factor-alpha (TNF) is known to inhibit fat cell development in vitro and to be expressed in adipose tissue suggesting that it may act as an auto-/paracrine regulator of adipose tissue mass in vivo. We demonstrate here that endogenous TNF-mRNA expression of cultured human preadipocytes and adipocytes is suppressed by the unspecific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), which is frequently used to trigger the differentiation process. As assessed by the measurement of glycerophosphate dehydrogenase, IBMX stimulated the differentiation of human preadipocytes in a dose dependent manner up to threefold but remained ineffective when cells were simultaneously treated with 1 nM TNF. These results suggest that the adipogenic effect of IBMX is mediated by suppression of endogenous TNF production.

Human milk and infant formula can induce in vitro adipocyte differentiation in murine 3T3-L1 preadipocytes.

The potential of infant diet to influence fat cell development has largely been examined in clinical studies with conflicting results. In this study, the direct effects of two standard infant formulas, Enfamil and Similac, as well as human milk were examined using a well characterized model of adipocyte differentiation, the 3T3-L1 murine preadipocyte cell line. After exposure to a hormonal regimen of insulin, dexamethasone, and 1-methyl-3-isobutylmethylxanthine, these cells undergo a mitotic expansion phase followed by terminal differentiation. On d 4 of hormonal exposure, greater than 95% of 3T3-L1 cells exhibit the morphologic and biochemical characteristics of mature adipocytes. In this study, cells were exposed to control medium, or control medium supplemented with either 10% Enfamil, 10% Similac, 10% human milk (skim or whole), or the standard hormonal regimen. Oil Red O-detectable lipid accumulation, immunocytochemical cell proliferation assays, and activated expression of adipocyte differentiation-specific mRNAs by Northern blot analysis were used to assess the effects of treatment on adipocyte differentiation. Results from each level of assessment revealed that both Enfamil and human milk were as effective as the standard hormonal regimen at stimulating adipocyte differentiation. In contrast, results from treatment with Similac or human skim milk were indistinguishable from control unstimulated cells. This study, demonstrating that Enfamil and human milk are capable of independently inducing in vitro adipocyte differentiation, suggests that diet during infancy could influence body fat development.

Expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARgamma is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARgamma is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARgamma expression in primary macrophages and monocytic cell lines. PPARgamma mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARgamma expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARgamma expression in macrophages. TPA induced the expression of PPARgamma in RAW 264.7 macrophages by increasing transcription from the PPARgamma1 and PPARgamma3 promoters. In concert, these observations provide insights into the regulation of PPARgamma expression in activated macrophages and raise the possibility that PPARgamma ligands may influence the progression of atherosclerosis.

The serpent gene is necessary for progression through the early stages of fat-body development

The serpent ( srp) gene, also known as ABF, codes for a GATA-like transcription factor and is involved in the transcriptional activation of Adh in the larval fat body or adipose tissue. Here, we describe the tissue-specific distribution of SRP protein in various stages of embryonic development and describe srp's role in early fat-cell development. SRP protein was detected in the progenitor fat-body cells and is present in the developing fat-body cells and in the mature embryonic fat body. An analysis of srp embryos revealed a gradual loss of precursor fat cells that is likely due to apoptosis. Within the fat-cell lineage, srp is necessary for progression through early stages of fat-cell development and may be involved in the transactivation of genes necessary for fat-cell differentiation.

Identification of fat-cell enhancer activity in Drosophila melanogaster using P-element enhancer traps.

To identify genes important in fat-cell metabolism and development, we have screened Drosophila stocks carrying an engineered transposable element that can reveal the presence of nearby enhancer elements. We have identified those "enhancer-trap lines" that contain transposable P elements integrated near fat-cell specific enhancer elements. We anticipate that the genes associated with these enhancers will provide information concerning fat-cell function and serve as target genes for studying fat-cell specific gene expression. Furthermore, the identification of enhancer-trap lines active in the developing fat cell should provide an entry point into the molecular and genetic analysis of early fat-cell development. Analysis of two lines has revealed that the transcription factors svp, a steroid-hormone receptor, and Kr, a zinc-finger protein, are present in the fat body; these factors are likely to be involved in fat-cell gene expression. In two other lines, beta-galactosidase was detected in a subset of adepithelial cells that may be the precursors to the adult fat cell. And finally, in a single line transgene activity is present in the progenitor cells of the embryonic fat body. The genes associated with these enhancer-trap lines may be involved in fat-cell development.

Norepinephrine utilizes alpha 1- and beta-adrenoreceptors synergistically to maximally induce c-fos expression in brown adipocytes

In order to examine how norepinephrine stimulates proliferation and differentiation in brown fat cells, we have investigated the ability of brown fat cells to respond to norepinephrine stimulation with an increase in the expression of the proto-oncogene c-fos. Stimulation of brown fat precursor cells (isolated from young mice and grown for 4 days in culture) with norepinephrine led to a marked but transient (maximal approximately 30 min) induction of c-fos expression. The magnitude of this induction was similar in pre- and postconfluent cells. The norepinephrine effect could be blocked by both alpha 1- and beta-adrenergic antagonists. Forskolin had a small inductive ability, as had the selective alpha 1-agonist cirazoline, but with both together a high induction was obtained. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) could in itself induce c-fos expression, but pretreatment with TPA did not abolish the ability of norepinephrine to induce c-fos expression, indicating that TPA-sensitive protein kinase C was not a primary mediator in this pathway. Also the Ca2+ ionophore A23187 had in itself an inductive ability, but A23187 in combination with forskolin led to a large increase in c-fos expression, indicating synergistic interaction between a cAMP pathway and a [Ca2+]i pathway. This interaction was not proximal, i.e. alpha 1 stimulation or increase in [Ca2+]i by A23187 did not augment forskolin-induced cAMP levels, and beta stimulation or forskolin did not affect [Ca2+]i levels; and it did not require protein synthesis. It was concluded that norepinephrine, in agreement with its fundamental role in the control of brown fat cell growth and development, was able to induce c-fos expression, that this induction was not exclusively linked to promotion of either proliferation or differentiation, and that the induction was mediated via a distal synergism between beta/cAMP and alpha 1/[Ca2+]i pathways, thus conferring to the alpha 1-adrenoreceptors on the cell a potentially significant role in the control of cell growth and development.

Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes.

Murine 3T3-L1 preadipocytes proliferate normally in medium containing fetal calf serum depleted of insulin, growth hormone, and insulin-like growth factor-I (IGF-I). However, the cells do not differentiate into adipocytes in the presence of the hormone-depleted serum. Supplementation of the growth medium with 10-20 nM IGF-I or 2 microM insulin restores the ability of 3T3-L1 cells to develop into adipocytes. The cells acquire an adipocyte morphology, accumulate triglycerides, and express a 450-fold increase in the activity of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. The increase in glycerol-3-phosphate dehydrogenase activity is paralleled by the accumulation of glycerol-3-phosphate dehydrogenase mRNA and mRNA for the myelin P2-like protein aP2, another marker for fat cell development. IGF-I or insulin-stimulated adipogenesis in 3T3-L1 cells is not dependent on growth hormone. Occupancy of preadipocyte IGF-I receptors by IGF-I (or insulin) is implicated as a central step in the differentiation process. The IGF-I receptor binds insulin with a 70-fold lower affinity than IGF-I, and 30-70-fold higher levels of insulin are required to duplicate the effects of an optimal amount of IGF-I. The effects of 10-20 nM IGF-I are likely to be mediated by high affinity (KD = 5 nM) IGF-I receptors that are expressed at a density of 13,000 sites/preadipocyte. In undifferentiated cells the IGF-I receptor concentration is twice that of the insulin receptor. After adipocyte differentiation is triggered, the number and affinity of IGF-I receptors remain constant while insulin receptor number increases approximately 25-fold as developing adipocytes become responsive to insulin at the level of metabolic regulation. Thus, preadipocytes have the potential for a maximal response to IGF-I, whereas the accumulation of more than 95% of adipocyte insulin receptors and the appearance of responsiveness to insulin are consequences of differentiation. IGF-I or insulin is essential for the induction of a variety of abundant and nonabundant mRNAs characteristic of 3T3-L1 adipocytes.

Influence of genetic type, slaughter weight and sex on ovine muscle fiber and fat-cell development.

Histological properties from the longissimus (LD) and semimembranosus (SM) muscles of 51 wether and ewe lambs from Hampshire rams and two ewe genetic types (SR, 1/2 Suffolk and 1/2 Rambouillet, and FS, 1/2 Finnish Landrace and 1/2 Southdown) and three slaughter live weights (32, 41 and 50 kg) were compared. Fibers in both muscles were classified as beta R (red), alpha R (intermediate) or alpha W (white). All LD muscle fiber types from FS ewe lambs increased in diameter from 32 to 41 kg, but decreased in diameter from 41 to 50 kg. Also, this quadratic effect with slaughter weight was found in alpha R and alpha W fibers from the SM muscle of FS wether and ewe lambs. However, diameters of alpha R and alpha W fibers from SR wether and ewe lambs and beta R fibers from SR ewe lambs increased linearly in the SM muscle with increasing slaughter weight. As slaughter weight increased, the proportion of alpha R fibers decreased in both the LD and SM muscles for SR wether and FS wether and ewe lambs. Concurrent with the increase in slaughter weight and decrease in alpha R fiber percentage, the proportion of alpha W fibers increased in the LD muscle of SR wether lambs, the SM muscle of SR ewe lambs and both muscles of FS wether and ewe lambs. Genetic type and sex groups, except SR ewe lambs, support the theory of transformation of alpha R to alpha W fibers with increasing slaughter weights. Fat-cell diameters increased in both muscles of SR wether and FS ewe lambs with increasing slaughter weights. Increases in fat traits of lamb carcasses were related to increases in red-fiber size. Alpha red fiber numbers were inversely related (P less than .05) to alpha W fiber numbers in both the LD and SM muscles (r = -.83 and -.79). The proportion of alpha R to alpha W fibers might be used as an indicator of physiological maturity for lambs.

Adipose tissue development in the fetal pig after decapitation.

The effect of fetal decapitation on adipose tissue development in utero was studied in the pig. Pig fetuses were decapitated at 45 days of gestation, and dorsal subcutaneous adipose tissue was analyzed at 110 days of gestation. The histology, histochemistry, ultrastructure, size and lipoprotein lipase activity of adipocytes from decapitated and sham-decapitated control fetuses were determined. Decapitation resulted in a poorly developed dermis and epidermis, a poorly developed outer layer of adipose tissue and larger fat cells that were metabolically and structurally more mature. Fewer fat cell clusters in decapitated fetuses were associated with fewer blood vessels in the subcutaneous layers. The results of this study demonstrate that fat cell development in subcutaneous tissues may be an integral aspect of the development of skin and associated structures.