The development of adipocytes in mice and humans follows a defined pathway that begins with a common MSC that is pluripotent and is referred to as adipogenesis [78
]. The early steps of the pathway leading to the generation and the commitment of MSCs to the adipocyte lineage is unknown. Hypothetically, the determination of the fate of MSCs occurs early in the stages of cell differentiation (“commitment”) and involves the interplay of intrinsic (genetic) and environmental (local and systemic) conditions to ultimately define cell fate. Factors such as those discussed earlier that determine MSC proliferation and differentiation also govern early adipocyte development and function. Currently, however, little is known about this process- from MSC to preadipocyte differentiation. The steps governing the process from preadipocyte to adipocyte differentiation however, have been well defined.
During adipogenesis MSC-derived or preadipocytes differentiate into lipid-laden and insulin-sensitive adipocytes [79
]. An overview of the stages of adipogenesis is presented in . The acquisition of adipocyte phenotype and development of adipocyte function is characterized by chronological changes in the expression of multiple genes. This process is highly regulated by the appearance of early, intermediate and late mRNA/protein markers and triglyceride accumulation ( and ).
Figure 6 Molecular mechanisms underlying adipocyte dysfunction in a hypertrophied adipocyte. Hyperglycemia results in the increase of ROS production within the mitochondria via a number of mechanisms including a reduction in the glutathione/glutathione disulfide (more ...)
Figure 7 The effect of HO-1 and HO-2 siRNA on adipogenesis. Lipid droplets area was determined by Oil red O staining after 14 days. * p<0.01 vs. control and #p<0.001 vs. HO-1 siRNA. Each bar represents means ± SE of 5 independent experiments. (more ...)
Briefly, the stages of adipocyte differentiation are: (1) MSC-derived adipocyte growth arrest; (2) clonal expansion, (3) a second stage of growth arrest or early differentiation and (4) terminal differentiation- development of mature adipocyte phenotype [80
]. Concurrently with the development of a mature adipocyte, there is an increase in transcription or de novo
expression of several genes including- Glut 4, insulin receptor, fatty acid synthase (FAS), [80
]. In the early phase of differentiation, preadipocyte cells are morphologically similar to fibroblasts. After clonal expansion, continued induction of adipogenesis leads to a drastic change in cell shape. Preadipocytes convert to a spherical shape, lipid droplets accumulate and the preadipocyte progressively acquires the morphological and biochemical characteristics of a mature adipocyte followed by triglyceride accumulation [81
PPARγ and c/EBPα are central components of this network and are critical factors that initiate a cascade of other factors that enhance differentiation of adipocytes. A secondary level of control exists, in which recruited coactivators and corepressors ultimately define the phenotype and metabolic fate of differentiated adipocytes.
PPARs are part of the steroid/retinoid nuclear hormone receptor superfamily and include isoforms α, γ, and δ [78
]. PPARs play a role in cellular development, differentiation, and metabolism. PPARγ, specifically, is crucial for adipogenesis and is necessary [82
], along with C/EBPα, for adipocyte differentiation. PPARγ for differentiation, survival and metabolism. PPARγ is required for the maintenance of adipocyte differentiation. In differentiated 3T3L-1 cells, dominant-negative PPARγ expression led to dedifferentiation with a resultant loss of lipid accumulation and a decreased expression of adipocyte markers [83
]. Additionally, the deletion of PPARγ within the germline renders these cells non-viable.
PPARγ can be activated by both endogenous and exogenous synthetic ligands. Endogenous ligands of PPARγ are largely unknown, but several lipid metabolites have been implicated including polyunsaturated fatty acids and eicosanoids [79
]. Synthetic ligands include thiazolidinediones (TZDs), a class of widely used drugs that are known PPARγ agonists, that act specifically to increase insulin sensitivity. TZDs increase insulin sensitivity by stimulating muscle glucose disposal and inhibiting hepatic glucose output. There are three plausible mechanisms that could explain how the activation of PPARγ acts as a systemic regulator of insulin sensitization. Firstly, pharmacological activation of PPARγ leads to adipose expansion through adipocyte hyperplasia, increasing the number of new preadipocytes. These adipocytes are small in size and able to effectively store lipids, thereby reducing lipotoxicity in the muscle and liver and release of adiponectin [84
]. This process involves activation of the genes encoding molecules that promote lipid storage and lipogenesis, i.e.
aP2 (fatty-acid binding protein), CD36 (receptor for lipoproteins), and FATP-1 (fatty acid transporter) [85
]. Activation of these genes leads to repartitioning of lipids resulting in increased triglyceride content of adipose tissue, lowered free fatty acid content in circulation and availability for liver and muscle use, thereby improving insulin sensitivity. Secondly, PPARγ agonists act by inhibiting the expression of inflammatory cytokines including TNF-α which promote insulin resistance [86
]. Thirdly, PPARγ agonists stimulate the production of adiponectin, which promotes fatty acid oxidation and insulin sensitivity in muscle and liver [85
], thereby decreasing glucose output by the liver and increasing glucose usage by the muscle. Thus, PPARγ is necessary for induction of adipogenesis, maintenance of the adipocyte lineage, and to act as a systemic regulator of insulin sensitivity and adipokine production.
The CCAAT enhancer binding proteins (C/EBPs) belong to the basic-leucine zipper class of transcription factors which are all expressed during adipogenesis [80
]. C/EBPβ and C/EBPδ are expressed early during adipogenesis and promote the main regulators of terminal adipocyte differentiation, PPARγ and C/EBPα. The interaction between PPARγ and C/EBPs establishes the mature state of adipocytes with all of their appropriate and necessary functions. The sequence of events is as follows: an increase in C/EBP-β above a threshold level induces expression of PPAR-γ. Upon ligand activation, PPAR-γ, in concert with C/EBP-α, leads to full adipocyte differentiation [81
]. After PPARγ stimulation, fat cells lacking C/EBPα, are able to accumulate lipid, express most adipogenic markers, but have poor insulin sensitivity due to lower levels of insulin receptor and low insulin receptor substrate-1 (IRS-I) production [80
]. In addition, insulin-dependent glucose uptake is also completely absent, demonstrating a crucial role for C/EBPα in regulation of insulin sensitivity. Taken together, current data demonstrates the necessity of both C/EBPα and PPARγ in not only inducing development of adipocytes but also their importance in establishing adipocyte function; i.e. glucose uptake and insulin sensitivity ().
The physiological stimuli that control MSC differentiation in vivo are largely unknown. However, induction of adipogenesis in vitro
requires a “differentiation cocktail” containing high glucose, insulin, glucocorticoids, and additional factors such as dexamethasone, methylisobutylxanthine and/or indomethacin. These induce the specific signaling cascades necessary to switch the genetic programming from preadipocytes (or stem cell fate) to mature adipocyte mode. Insulin is a required regulator of both early and late adipogenesis. In the early stages of adipogenesis, insulin mediates its actions through insulin growth factor-1 receptor signaling, and later through insulin receptor as insulin receptors increase during the late stages of adipogenesis. Further down the insulin cascade, inhibition of phosphatidylinositol-3-kinase (PI3K) as well as loss of AkT
1 or 2/protein kinase B (PKB) represses adipogenesis [87
]. The insulin signaling cascade eventually leads to activation, directly and indirectly through blockade of repressors of C/EBPα and PPARγ. Insulin also promotes glucose uptake in both muscle and adipose tissue as well as triglyceride catabolism [85
], regulating the normal metabolic functions of the mature adipocyte. Glucocorticoids are part of the nuclear hormone family, and their actions are not well characterized in adipocyte differentiation. However, in vitro
studies have shown that glucocorticoid treatment is either required for differentiation or acts to accelerate the process. In 3T3L-1 cells, this action is activated through induction of C/EBP-δ [82
]. Methylisobutylxanthine (MIX), an inhibitor of phosphodiesterases, increases intracellular cAMP, activating adipocyte differentiation in a PKA- independent manner [88
]. MIX has also been shown to increase the expression of C/EBP-β which is required for the subsequent expression of PPAR-γ [81
]. Similarly, indomethacin acts as an upstream regulator of PPARγ2, by increasing the expression of C/EBP-β [89
Although factors controlling adipogenesis in vivo
are largely unknown, there are a number of candidates that mediate adipocyte differentiation in culture (in vitro
) and are thought to control adipocyte accumulation and function in vivo
. Two main factors fit this criterion: (1) high glucose and (2) ROS. They have been implicated as the link between adipogenesis and metabolic diseases such as T2DM. As described earlier, recent studies have demonstrated induction of oxidative stress by high glucose (through three mechanisms, NAD(P)H oxidase, xanthine oxidase, and mitochondrial respiratory chain) is associated with diabetic complications [90
]. Therefore, these factors may lead to adipocyte differentiation associated with adipocyte dysfunction and formation of adipocytes external to normal adipocyte depots i.e. muscle, liver, and pancreas leading to advanced diabetic complications.
How do ROS and high glucose lead to adipocyte dysfunction and insulin resistance? Firstly, we must define what adipocyte function is. Adipose tissue is a key endocrine organ whose functions include (1) lipid uptake, storage, and synthesis; (2) secretion of endocrine, paracrine, and autocrine factors that regulate insulin sensitivity and glucose uptake; and (3) secretion of anti-inflammatory molecules such as adiponectin, IL-1, and IL-10 (). Critically, increases in adipocyte size result in decreased secretion of adiponectin, and increased secretion of inflammatory molecules such as MCP-1, IL-6, and TNF-α that have systemic effects on other tissues including, vasculature, liver, muscle, and β cells.
Overview of stages of adipocyte differentiation. PPAR-y, peroxisome proliferator-activated receptor-y; C/EBP,CCAAT/enhancer binding protein).
Adipokines, are highly diverse in terms of structure and physiologic function and are involved in the regulation of blood pressure (angiotensinogen), vascular haemostasis (PAI-1), lipid metabolism e.g. (retinol binding protein, cholesteryl ester transfer and angiogenesis (VEGF)). The release of these cytokines by adipocytes has led to the conclusion that adipose tissue has many systemic effects which are altered during adipocyte hypertrophy and obesity. The most studied adipokines include adiponectin, acrp30, which is exclusively secreted from adipose tissue and is known for its role in insulin sensitivity, and the pro-inflammatory cytokines TNF-α, IL-6, IL-1β, IL-8, IL-10, and MCP-1 that are released during adipocyte hypertrophy and obesity [91
In summary, overnutrition (excessive amounts of FAs) and high glucose produces a hypertrophied adipocyte resulting in detrimental perturbations in both mitochondrial and ER function, both of which undergo cellular changes that result in the increased generation of ROS and activation of an inflammatory cascade. This culminates in the release of inflammatory mediators that are associated with insulin resistance and negative downstream effects ().
Systemically, increases in ROS are related to the production of inflammatory cytokines, obesity and insulin resistance. As discussed earlier, induction of HO-1 decreases superoxide and ROS generation, resulting in an increase in adiponectin and a subsequent elevation in tolerance to ROS in the heart [93
]. An inverse relationship between levels of ROS and adiponectin exists and was demonstrated in an elegant study that encompassed patients, cultured adipocytes and obese mouse models. Fat accumulation was closely correlated with markers of systemic oxidative stress [94
]. In addition, plasma adiponectin levels correlated inversely with systemic oxidative stress. In cultured adipocytes, the oxidative stress suppressed mRNA expression, secretion of adiponectin and increased IL-6 and MCP-1 mRNA expression [34
]. Furthermore, treatment with the NADPH oxidase inhibitor, apocynin, reduced oxidative stress in WAT and increased plasma adiponectin levels in KKAy
]. These results indicate that a local increase in oxidative stress in accumulated fat caused dysregulated production of adipokines. The down regulation of adiponectin expression was partially attributed to reduction of nuclear PPARy gene expression under conditions of oxidative stress [94
Induction of HO-1 modulates metabolic syndrome, obesity, and insulin resistance [1
]. The changes that are observed after increased expression of HO-1 in obese and diabetic animal models include: (1) prevention of weight gain (2) reduction of inflammatory cytokines levels (3) restoration of normal insulin sensitivity and (4) improved vascular reactivity. Taken together, these are the major components of metabolic syndrome and possible mechanisms by which each of the parameters are modified by HO.