In humans, unlike other organs such as the liver, heart, or lung, the AT does not have a well-defined tissue demarcation. Fat under the skin or dermis is referred to as subcutaneous AT, whether it lies under the skin of the abdomen, extremities, or other parts of the body. Fat found in the visceral cavity of the body is largely referred to as intraabdominal or omental AT. In humans, the subcutaneous (sc) and the omental forms are the two large AT depots that have been studied in detail, whereas smaller AT depots, such as those behind the eyes (retro-orbital), knees (periarticular), around the hip joints, or beneath the skull, have received little attention. It is unclear whether, in addition to these differences in the anatomical location of AT, there are physiological differences as well between these AT depots. Assuming that AT in the different anatomical sites has similar functions would be an oversimplification. Unfortunately, little is known about the physiological differences between the various anatomically distinct AT depots. Some investigators consider the two large depots, sc and intra-abdominal, as metabolically active, whereas the smaller depots, such as retro-orbital, periarticular, or beneath the skull, are seen as relatively inert or mechanical AT (57). Others propose that these so-called mechanical ATs, which are intertwined with the lymphatic system, participate in the immune response via paracrine mechanisms (58,59). Deletion of the mouse Prox-1 gene, which is expressed only in the lymphatic system, revealed the connection between lymphatic system and AT when these animals developed obesity (60,61).
Much of our understanding of AT biology has been derived by analyzing the mouse 3T3-L1 fibroblast cell line, which has been used extensively to dissect the mechanisms of adipocyte differentiation and maturation using an in vitro cell culture model system. As shown in Fig. 5A, the initial phase begins with the recruitment of mesenchymal stem cells to committed cell lineages: osteoblasts, preadipocytes, chondrocytes, and myoblasts. Although not much is known about the differentiation of these cell lineages, recent studies show that transcription factors such as TAZ (transcriptional coactivator with PDZ-binding motif) are among the very first transcription factors contributing toward the genesis of preadipocytes from undifferentiated mesenchymal cells (62), with the acquisition of the cell surface markers CD34+/CD31- (63). Based on the deletion of AGPAT2 and BSCL2 genes in lipodystrophy, these genes could also affect the formation of preadipocytes.
Much is now known regarding the differentiation of preadipocytes to adipocytes, as described below. The capacity of mature adipocytes for enhanced lipogenesis is predominantly mediated by the activation of the master transcription factor SREBP-lc
Fig. 5. Schematics for the differentiation and maturation of adipocytes from mesenchymal stem cells. (A) Mesenchymal stem cells, by a mechanism that is still not well understood, differentiate into cell lineages, including preadipocytes. A transcriptional coactivator with PDZ binding motif (TAZ) when downregulated increases the number of committed preadipocytes and acquire the cell surface markers such as CD34+/CD31- (63). Shown also are AGPAT2 and BSCL2 which could also affect this step of pre-adipocyte differentiation. These are then further differentiated into mature adipocytes by serial activation of the gene program, as shown in (B). Genetic evidence obtained from lipodystrophic patients, AGPAT2 and BSCL2, could affect this step as well. Mature adipocytes accumulate lipids owing to activation of transcription factors such as SEBRP-lc, which induces expression of all lipogenic enzymes and the activation of triglyceride-synthesizing genes. Mature adipocytes are engorged with lipid accumulation, enhanced lipogenesis, or return to the original cell size because of increased lipolysis, mainly owing to activation of hormone-sensitive lipases. The least understood step is adipocyte disintegration, most likely via activation of caspases-8 (apoptosis) (93) or additional unknown factor(s). (B) The main transcriptional events essential for differentiation of the preadipocytes to adipocytes are described in the text. Shown in the box are adipocyte inhibiton factors including Pref-1, FOXO1, FOXA2, members of Wnt (WntlOb) and notch (Notch/ HES-1) signaling pathway, which must be downregulated for the differentiation of preadipocytes to adipocytes to occur. Recent experiments have discovered additional factors necessary for the differentiation and maturation of the adipocytes. Shown are the transcription factor, Krox20, which is upregulated and, in turn, increases the expression level of C/EBP-P and -6. Transcription factor KLF5 is shown to up regulate PPARy. The level of PPARy is also regulated by AKT2. The transcription factors GATA-2 and -3 downregulate PPARy and C/EBP-a and -p. Proteins such as KSR1 inhibition negatively regulate adipogenesis. Expression of cdk4 activates E2F, a transcription factor, which facilitates expression of PPARy via downregulation of retinoblastoma (RB) protein. Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor P superfamily, has a role in terminal differentiation of preadipocytes to adipocytes. Although a nuclear receptor
(64), which induces the expression of all lipogenic enzymes. Mature adipocytes cycle between stages of lipid accumulation during the fed state and lipolysis during fasting. It is unclear if aged adipocytes ultimately disintegrate via apoptosis or remain quiescent in a lipid-depleted state.
Many studies have established specific hormones, transcriptional factors, and intra-cellular signals that participate in the differentiation of preadipocytes to adipocytes. Treatment of 3T3-L1 cells with adipogenic factors, including dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum (Fig. 5B), sets in motion a cascade of events resulting in the upregulation of transcription factors such as the CCAAT/enhancer binding protein (C/EBP-P and -8) (65), which then induce expression of the C/EBP-a and PPARy transcription factors (66). Simultaneously, several transcription factors are downregulated as well, shown in the box in Fig. 5B. These include the well-known adipogenesis inhibition factor, preadipocyte factor (Pref)-1 (67), FOXO1, FOXA2, members of Wnt (Wnt10b) and notch (Notch/HES-1) (68,69) signaling pathways, which are downregulated during the differentiation of preadipocytes to adipocytes (70). The importance of the nuclear receptor PPARy for adipogenesis is well documented, as overexpression in 3T3-L1 cells is sufficient for differentiation to adipocytes. In addition, other nuclear receptors, including nerve growth factor-induced gene B (NGF1-B), nuclear receptor-related factor 1 (NURR1), neuron-derived orphan receptor 1 (NOR1), and vitamin D receptor (VDR), are upregulated within the first 24 h of initiation of adipogenesis. After 48 h, the expression of other nuclear receptors including PPARy, liver X receptor (LXR) a, retinoic acid-related orphan receptor (ROR)y retinoic acid X receptor y (RXRy), and androgen receptor (AR) rise; these factors remain elevated for 15 d (71).
Recent experiments have discovered additional factors necessary for the differentiation and maturation of adipocytes. The increased expression of a zinc-finger transcription factor, Krox20 (72), in turn increases the levels of C/EBP-P and -8. C/EBP-P and -8 induce the expression of C/EBP-a, another zinc-finger transcription factor, Kruppel-like transcription factor (KLF5) (73), and PPARy. Mouse embryonic fibroblast cells obtained from homozygous deletion of Akt2 had impaired adipocyte differentiation and downregulation of PPARy, suggesting that Akt2, a serine/threonine kinase, also activates expression of PPARy (74). Other zinc-finger transcription factors, such as GATA-2 and -3, are shown to downregulate PPARy (75), as well as C/EBP-a and -P, which could help explain lack of AT development in those depots that have increased expression of these transcription factors. Cell cycle regulators such as cyclin-dependent kinase 4 (cdk4) (76) may also facilitate the expression of PPARy. Bone morphogenetic protein 2 (BMP2) (77), a member of the transforming growth factor-^ superfamily, contributes to terminal differentiation of preadipocytes to adipocytes. Scaffolding proteins such as kinase suppressor of Ras 1 (KSR1) negatively regulate adipogenesis (78).
Fig. 5. (Continued) such as PPARy is well studied for adipogenesis, other nuclear receptors such as NGF1-B, NURR1, NOR1, and VDR are also upregulated within the first 24 h of initiation of adipogenesis. The increase in expression of nuclear receptors, including PPARy, LXRa, RORy, RXRy, and AR, occurs around 48 h after stimulation and remain elevated until 15 d. The interaction of many of these transcription factors, kinases, ligands, and metabolites may provide a better explanation for differential loss of adipose tissue.
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