Adipogenesis and Obesity

CHAPTER 2 1

Adipogenesis and Obesity Josue Moura Romao, Le Luo Guan Department of Agricultural, Food and Nutritional Science, University of Alberta

CONTENTS Glossary��������������������������������������������������������������������������������������������������� 540 21.1 Adipose Tissue������������������������������������������������������������������������������ 540 21.1.1 Triacylglycerol Synthesis and Storage��������������������������������������������������541 21.1.2 Lipolysis and Free Fatty Acids Release������������������������������������������������541 21.1.3 Adipokine Release����������������������������������������������������������������������������������541

21.2 Adipogenesis��������������������������������������������������������������������������������� 542 21.2.1 Commitment�������������������������������������������������������������������������������������������543 21.2.2 Differentiation�����������������������������������������������������������������������������������������544

21.3 Obesity�������������������������������������������������������������������������������������������� 545 21.3.1 Etiology����������������������������������������������������������������������������������������������������545 23.3.2 Health Concerns�������������������������������������������������������������������������������������545

21.4 miRNA Regulation of Adipogenesis��������������������������������������������� 546 21.4.1 miRNAs in Adipocyte Commitment������������������������������������������������������547 21.4.2 miRNAs in Adipocyte Differentiation���������������������������������������������������548

21.5 miRNAs in Obesity������������������������������������������������������������������������ 552 21.5.1 Mouse Studies�����������������������������������������������������������������������������������������553 21.5.2 Human Studies����������������������������������������������������������������������������������������554

21.6 Factors That Influence miRNA Expression in Adipose Tissue������������������������������������������������������������������������������ 555 21.6.1 Diet�����������������������������������������������������������������������������������������������������������556 21.6.2 Adipose Depots���������������������������������������������������������������������������������������557

21.7 Conclusions and Future Perspectives������������������������������������������ 559 Chapter Questions���������������������������������������������������������������������������������� 561 Short-Answer������������������������������������������������������������������������������������������������������561

References����������������������������������������������������������������������������������������������� 562 Online Resources������������������������������������������������������������������������������������ 564 Further Reading�������������������������������������������������������������������������������������� 565 MicroRNA in Regenerative Medicine. http://dx.doi.org/10.1016/B978-0-12-405544-5.00021-6 Copyright © 2015 Elsevier Inc. All rights reserved.

539

540

CHAPTER 21:  Adipogenesis and Obesity

GLOSSARY Adipocyte commitment  the process by which stem cells are converted into preadipocytes. Adipocyte differentiation  the conversion of preadipocytes into mature adipocytes. Adipogenesis  the development of fat cells (adipocytes) from nonspecialized cells. Adipokine  a cytokine produced by adipose tissue (e.g., leptin and adiponectin). BMI (body mass index)  a measure of weight status and uses the formula of body mass divided by height squared (kg/m2). Transfection  the introduction of nucleic acid into cells (e.g., siRNAs).

21.1 ADIPOSE TISSUE Adipose tissue, a specialized type of connective tissue, consists of a number of different cell types such as adipocytes, endothelial cells, and macrophages. It was traditionally recognized for its ability to store excess energy and to provide insulation and padding to the body. However, now adipose tissue is also seen as an endocrine organ responsible for secreting various enzymes, growth factors, matrix proteins, hormones, cytokines, and complement factors [1]. There are two types of adipose tissues in mammals, white adipose tissue (WAT) and brown adipose tissue (BAT), which differ morphologically and functionally. In a nonobese human, WAT is estimated to weigh 10–15 kg; it specializes in energy storage in the form of triacylglycerols, the major energy reserves for mammals. The release of triacylglycerols, which is called lipolysis, provides fuel to other organs. These properties make WAT an important regulator of energy homeostasis. Moreover, WAT performs other important processes, such as gluconeogenesis and lipoprotein synthesis [2]. BAT can dissipate energy through adaptive thermogenesis, leading to heat production [3], which is important to newborns as they lack the ability to shiver. The amount and disposition of lipids in WAT and BAT cells are also different. White fat cells generally have a major lipid droplet occupying most of the cytoplasm (unilocular), while brown fat cells have various small lipid droplets (multilocular) [3]. Differences in mitochondria amount, vascularization, and lipid content lead to the color difference between WAT and BAT [3].

SECTION KEY POINT Adipose tissue is a dynamic organ that plays a fundamental role in energy balance and performs

endocrine functions through the release of adipokines.

21.1  Adipose Tissue

21.1.1 Triacylglycerol Synthesis and Storage Most of the lipids stored in adipose tissue are triacylglycerols consisting of three molecules of fatty acids and one molecule of glycerol. The synthesis and storage of triacylglycerol in adipose tissue may utilize fatty acids derived from de novo lipogenesis or from those in a diet that is considered the main source of lipids [2]. De novo lipogenesis entails the synthesis of fatty acids from substrates that are not lipids, such as carbohydrates, including glucose as the main substrate [2]. The disposal of glucose after meals is directed mainly to muscle and adipose tissue; it happens under the influence of insulin, which is responsive to glucose levels in the blood, which rise after meals. The glucose supply can be directed to de novo fatty acid synthesis in the liver and adipocytes; in adipocytes it is stored as triacylglycerols. The fatty acids provided with the meals are usually absorbed by the intestine and then released to the bloodstream as chylomicrons, while the triacylglycerides, produced in the liver, are released to bloodstream as very low-density lipoproteins. Both can be hydrolyzed by lipoprotein lipase, and released free fatty acids can be taken up by adipocytes for storage in the form of triacylglycerols [2].

21.1.2 Lipolysis and Free Fatty Acids Release Intracellular lipolysis inside adipocytes entails a coordinated catabolism of triacylglycerols from lipid droplets, which are eventually broken down to form one molecule of glycerol and three molecules of unesterified fatty acids, which can be released. This is a sequential process that is performed by triglyceride hydrolase enzymes: hormone-sensitive lipase, adipose triglyceride lipase, and monoglyceride lipase—all of which are necessary for adequate hydrolysis of triglycerides, diglycerides, and monoglycerides, respectively [2,4]. Other factors can influence lipolysis, such as perilipins and potentially some lipid-droplet surface proteins that regulate this process by exposing or protecting the triglycerides in the core of the droplet from the action of lipases [4]. Free fatty acids are generally released from adipocytes during fasting periods as an important energy source for various organs, playing an important role in glucose homeostasis. Those circulating in the bloodstream promote the reduction of glucose uptake by adipocytes and muscles, and they increase the hepatic production of glucose. This strategy saves the carbohydrates for neurons and red blood cells that are dependent on this fuel, while it uses the lipid reserves to power other tissues [5]. Lipolysis is under the control of endocrine regulation, in which insulin acts as an anti-lipolytic factor, but is promoted by catecholamines [4]. It may also vary according to other factors, such as species, age, sex, location of fat depots, and development [4].

21.1.3 Adipokine Release The discovery of regulatory factors released by adipose tissue has increased over the years, and today several adipokines are known to be involved in

541

542

CHAPTER 21:  Adipogenesis and Obesity

different biological processes; these include adiponectin, apelin, chemerin, leptin, omentin, resistin, retinol-binding protein 4, tumor necrosis factor α (TNF-α), vistatin, vaspin, and others whose physiological functions have yet to be completely established [5,6]. Leptin is the most well studied adipokine and one of the first signaling molecules detected in adipocytes [6]. It represses food intake and promotes energy expenditure by targeting hypothalamic cell populations, inducing anorexigenic factors, and inhibiting orexigenic neuropeptides [5,7]. The leptin level in circulating serum is directly correlated with the total fat mass of the individual, positively correlated with insulin, and negatively correlated with glucocorticoid levels. Its synthesis is mainly controlled by the ingestion of food and by eating-related hormones, but its production can also be dependent on factors such as energy status and sex [7]. Adiponectin is a hormone produced by WAT, with a high concentration in the plasma; contrary to other adipokines, its levels are negatively correlated with fat mass. Therefore, obesity promotes reduction in adiponectin levels while weight loss increases them [5,6]. Adiponectin stimulates fatty acid oxidation and glucose uptake in muscle and adipose tissue [6]. It inhibits glucose production in the liver, and it has been implicated in cardiovascular health [7]. Resistin is a peptide produced by adipocytes in mice, but in humans it is produced by mononuclear cells, such as macrophages from adipose tissue and other locations. It has been reported to impact glucose metabolism by reducing insulin action in tissues such as liver and muscle, and it seems to be involved in a series of biological processes contributing to insulin resistance; it may also be involved in inflammatory, endocrine, and tumor diseases [8]. Omentin is a depot-specific adipokine that is produced by stromal vascular cells located in the visceral fat depot. It affects glucose uptake and works as an insulin sensitizer. Omentin alters the gastrointestinal barrier function, which is regulated by insulin and glucose levels [1,5]. TNF-α was the first adipokine to be linked to obesity, inflammation, and diabetes. Studies show that it ­influences energy homeostasis, inhibits insulin signaling in adipose tissue and liver, and influences adipose tissue function and expansion. The insulin resistance promoted by TNF-α happens by suppression of proteins involved in the insulin-dependent intake of glucose and by repression of PPARγ (peroxisome proliferator-activated receptor-γ) expression [1,6].

21.2 ADIPOGENESIS Adipogenesis is the process by which nonspecialized stem cells turn into mature adipocytes, as shown in Figure 21.1. It takes place in two stages: commitment and differentiation. In commitment, the first stage, stem cells are converted into preadipocytes. In differentiation, preadipocytes are converted into mature adipocytes that are capable of secreting adipokines and transporting,

21.2 Adipogenesis

FIGURE 21.1  Adipocyte commitment and differentiation in cell culture. MSCs (present in the vascular stroma of adipose tissue) are multipotent cells able to commit not only to preadipocytes but also to pre-myoblasts, pre-chondrocytes, and pre-osteoblasts [9]. After they commit to form preadipocytes, they proliferate until growth arrest by contact inhibition. Differentiation is triggered by adequate adipogenic stimuli (hormonal induction), causing preadipocytes to be converted into mature lipid-assimilating adipocytes.

synthesizing, and releasing lipids. Our understanding of how adipogenesis works in mammals relies heavily on in vitro culture systems using preadipocyte cell lines, mature adipocyte-derived dedifferentiated fat cells, mesenchymal stem cells (MSCs), and adipose-derived stem cells [9].

SECTION KEY POINT Adipogenesis is a molecularly regulated process in which adipose stem cells are converted into mature

adipocytes through commitment and differentiation.

21.2.1 Commitment Commitment is the process in which pluripotent stem cells (PSCs) located in the vascular stroma of adipose tissue respond to signal(s) telling them convert

543

544

CHAPTER 21:  Adipogenesis and Obesity

into preadipocytes. Adipose lineage cells originate from PSCs of mesodermal origin. In the vascular stroma of fat tissue in mammals, two types of adipocyte precursors are found: pluripotent fibroblasts (stem cells) and unipotent preadipocytes. Once pluripotent fibroblasts commit to the adipose lineage, they can be induced only to form adipocytes and no other cell type. Bone morphogenic proteins (BMPs) play an important role in the commitment of PSCs into preadipocytes [10]. Both BMP2 and BMP4 were shown to induce commitment of C3H10T1/2 pluripotent stem cells into adipocytes. Treatment with these BMPs followed by exposure to differentiation inducers allows nearly all cells to enter the adipose development pathway, express specific adipocyte markers, and acquire the adipocyte phenotype [10]. Other factors that may play a role in stem cell commitment to the adipose lineage are cell shape and density. For example, plating human mesenchymal stem cells (hMSC) at low density favored the commitment to osteogenic lineage; high-density plating favored the commitment to the adipose lineage. Regarding cell shape, the hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while cells not allowed to spread assumed a round shape and became adipocytes [11].

21.2.2 Differentiation Differentiation is the process by which preadipocytes (less specialized cells) turn into mature adipocytes (more specialized cells). In cell culture models, in order to start differentiation preadipocytes must reach confluence (cell‒cell contact) and become growth-arrested (i.e., the cell cycle stops at the G0–G1 boundary), which is due to cell density inhibition. However, it is not the confluence but the growth arrest itself that is necessary for preadipocyte differentiation [12]. After confluence (cell–cell contact), when they receive adequate stimuli (via mitogenic and adipogenic inducers), preadipocytes synchronously restart the cell cycle and undergo mitotic clonal expansion. Mitotic clonal expansion consists of at least one round of cell replication and is a required step in the differentiation of preadipocytes into adipocytes in 3T3L1 cells [12]. It has been suggested that mitotic clonal expansion is needed to unwind the DNA, thus allowing transcription factor binding to genes involved in adipocyte differentiation [9]. The extensive change in cell shape from fibroblastic to spherical is considered as the first hallmark of adipogenesis. These changes happen simultaneously with changes in components and levels of extracellular matrix and cytoskeletal components. The modifications that the cells undergo are critical for adipogenesis regulation because they may promote the gene expression of C/EBPα (CCAAT/enhancer-binding protein α) and/or PPARγ. These are fundamental adipogenic transcription factors [12]. PPARγ and C/EBPα play an essential role in the progress of adipocyte differentiation because both act synergistically to activate the transcription of several genes that promote the adipocyte phenotype [13].

21.3 Obesity

21.3 OBESITY Obesity can develop in an individual when energy intake exceeds energy expenditure during a long period of time. It is considered a human epidemic, given that more than 1.5 billion people worldwide are overweight or obese [14]. In humans, obesity can be defined by the body mass index (BMI), the relationship between height and weight. According to this concept, a BMI between 18.5 and 25 kg/m2 is considered normal, while a BMI between 25 and 29.9 kg/m2 is overweight and over 30 kg/m2 is obese [15]. However these parameters are not absolute and other factors may be accounted for as well, such as ethnicity, age, and physical activity [15].

SECTION KEY POINT Obesity is the excessive accumulation of adipose tissue. This condition has a multifactorial etiology

and its consequences can be life-threatening.

21.3.1 Etiology Several factors contribute to obesity, such as inheritance (genetics). When a person has a mutation of the gene encoding leptin or its receptor [15,16], he or she may develop obesity. Environmental causes, such as social pressure and food marketing, may play a role in the physiological regulation of appetite [15]. In addition, there are high-energy diets that promote a reduced satiety response. Obesity can also be caused by low levels of physical activity. Modern lifestyles and the dramatic decrease in exercise have led to a great part of the population becoming sedentary [15]. Other factors, such as endocrine disease and drug use (e.g., anti-convulsants, neuroleptic agents, and insulin for diabetes), are less important, but may cause obesity in some people [15,16].

23.3.2 Health Concerns Obesity is a potentially life-threatening condition, being associated with a number of disorders [17,18] (Figure 21.2). For instance, cardiovascular diseases (e.g., hypertension) have been reported to be five times more prevalent in obese individuals than in those with normal weight. The increase in high blood pressure rates is partially due to the elevated release of angiotensinogen from adipocytes [19]. Also, the onset of type 2 diabetes is highly correlated with the obesity condition [19]. Certain cancers also may be related to obesity. It is reported that 10% of cancers in nonsmokers are related to obesity, and that 25–35% of breast, colon, endometrium, and esophagus cancers are due to inactivity and overweight. Besides these disorders, the obesity condition can be a promoter of many other problems, such metabolic syndrome, gall bladder

545

546

CHAPTER 21:  Adipogenesis and Obesity

FIGURE 21.2  Obesity and associated health risks.

disease, and detrimental effects on respiration and reproductive functions [15,19]. As described previously, fat tissue secretes various adipokines, and increases in fat mass can enhance the production of adipokines, which may trigger a series of disorders. For example, high levels of inflammatory peptides (TNF-α, IL-6, leptin, resistin, MCP-1, and PAI-1) produced in obese individuals favor insulin resistance and increase the risk of atherosclerosis [20].

21.4  miRNA REGULATION OF ADIPOGENESIS MicroRNAs (miRNAs) have been reported to regulate several biological processes, from embryonic development to diseases. It is estimated that over 60% of messenger RNAs are conserved miRNA targets [21]. Several studies have shown that miRNAs play an important role in adipogenesis [22]. They were first shown to regulate adipogenesis in a Drosophila study. In 2004, miRNA miR-143 was the first found to be involved in mammalian adipogenesis, and since then the involvement of a number of other miRNAs in adipogenesis have been reported, as shown in Table 21.1. To date, hundreds of miRNAs have been detected in the adipose tissue of different species [22]. This section explores the roles of miRNAs in commitment to the adipose lineage, the mechanism by which miRNAs enhance or inhibit adipocyte differentiation, and the regulation of miRNAs in adipose tissue.

SECTION KEY POINT miRNAs impact mammalian adipogenesis by regulating the expression of genes involved in

the commitment or differentiation of adipocytes leading to either pro- or anti-adipogenic effects.

21.4  miRNA Regulation of Adipogenesis

Table 21.1  miRNAs in Adipogenesis miRNA

Target

Function

Species

Cell Culture Model

let-7 miR-15a miR-17-92 miR-21 miR-27a/b miR-30a/d miR-30c miR-103 miR-130

HMGA2 DLK1 Rb2/p130 TGFBR2 PPARγ RUNX2 PAI1, ACVR1 PPARγ

Anti-adipogenic Pro-adipogenic Pro-adipogenic Pro-adipogenic Anti-adipogenic Pro-adipogenic Pro-adipogenic Pro-adipogenic Anti-adipogenic

Mice Mice Mice Human Human, mice Human Human Mouse Human

miR-138

EID1

Anti-adipogenic

Human

miR-141 miR-143

– ERK5

Pro-adipogenic Pro-adipogenic

Mouse Human

miR-155, mIR-221, miR-222 miR-199a miR-200a/b/c miR-204, miR-211

–

Anti-adipogenic

Human

SMAD1 – RUNX2

Pro-adipogenic Pro-adipogenic Pro-adipogenic

Mouse Mouse Mouse

miR-210 miR-369-5p

TCF712 FABP4

Pro-adipogenic Anti-adipogenic

Mouse Human

miR-371

–

Pro-adipogenic

Human

miR-375 miR-378 miR-429 miR-448

– – – KLF5

Pro-adipogenic Pro-adipogenic Pro-adipogenic Anti-adipogenic

Mouse Mouse Mouse Mouse

3T3-L1 3T3-L1 3T3-L1 hASCs 3T3-L1, OP9, hMADs hMADs hASCs 3T3-L1 Primary human preadipocytes Adipose mesenchymal stem cells ST2 Primary human preadipocytes Human mesenchymal stromal cells C3H10T1/2, ATDC5 ST2 C3H10T1/2, bone marrow stromal cells 3T3-L1 Human mesenchymal stromal cells Human mesenchymal stromal cells 3T3-L1 ST2, 3T3-L1 ST2 3T3-L1

miR-519

PPARα

Pro-adipogenic

Human

21.4.1 miRNAs in Adipocyte Commitment Multipotent stem cells from adipose tissue can commit to form adipocytes or to generate other lineages of tissues such as bones, cartilage, and muscle under the appropriate stimuli. Therefore, specific molecular guidance directing precursor cells to commit to the adipose lineage must occur in order to form adipocytes. In this context, miRNAs seem to play an important regulatory role. For example, mesenchymal stem cells (MSC) induced to differentiate into adipocytes have miR-204 expression. For example, when miR-204 was overexpressed, osteogenesis was inhibited while adipogenesis was promoted.

Primary human visceral preadipocytes

547

548

CHAPTER 21:  Adipogenesis and Obesity

On the other hand, when miR-204 expression was inhibited, osteogenesis was increased, and adipogenesis was impaired. This miRNA targets the runt-related transcription factor 2 (RUNX2), which is involved in osteoblastic differentiation [23]. miR-30a/d also targets RUNX2 and has been shown to induce adipogenesis when overexpressed [24]. Other studies reported miRNAs that impact the fate of stem cells from adipose tissue with direct impact on adipogenesis. The wingless-related MMTV integration site signaling pathway (WNT) can inhibit commitment into the adipose lineage, as observed in MSCs for which osteogenesis rather than adipogenesis was favored when they received ectopic expression of WNT10B, which is part of WNT signaling. miR-141, miR-200a/b/c, and miR-429 inhibited Wnt signaling and, when overexpressed, induced adipocyte differentiation in ST2 marrow stromal cells [22]. The number of miRNAs that influence adipogenic commitment by targeting WNT signaling may be, as one study found, 18 potential miRNAs repressing it and 29 favoring it in 3T3-L1 cells. One of these miRNAs was further analyzed (miR-210) and confirmed to inhibit WNT signaling by targeting TCF712, which induced increased adipogenesis in the cell culture [23]. Other miRNAs favor adipogenic commitment by repressing transforming growth factor beta (TGF-β) signaling; for example, miR-21 targets transforming growth factor beta receptor II (TGFBR2), thus repressing TGF-β signaling. miR-199a targets SMAD family member 1 (SMAD1), which is regulated by bone morphogenic protein 2 (BMP-2), a protein from the TGB-F signaling pathway [23]. It is clear that miRNAs play a role in committing multipotent stem cells to the adipose lineage, and this might be one of the factors influencing the amount of adipocytes in adipose tissue.

21.4.2 miRNAs in Adipocyte Differentiation miRNAs That Increase Adipogenesis

Pro-adipogenic miRNAs regulate adipocyte differentiation. Their regulatory mechanism may impact various pathways and different steps of the differentiation process. The direct regulatory impact of miRNAs can be assessed by measuring the repression of their targets. In adipocyte differentiation, miRNA regulation can be evaluated by various parameters, such as adipocyte numbers and sizes, intracellular accumulation of triglycerides, differentiation time, expression of adipogenic transcription factors, and expression of adipocyte markers. For example, clonal expansion is a key event during adipogenesis of 3T3-L1 cells and is affected by miRNA regulation. miR-17–92, a cluster that includes miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b, and miR-92-1, is up-regulated at the clonal expansion stage during 3T3-L1 cell adipogenesis. These miRNAs repress the expression of the tumor suppressor protein Rb2/p130, which is part of the “p130:p107” switch,

21.4  miRNA Regulation of Adipogenesis

an important step in the progression of mitotic clonal expansion. When the miR-17–92 cluster is overexpressed, adipocyte differentiation is accelerated and triglyceride accumulation is increased [22]. In cell culture, miR-30c accelerated adipocyte differentiation in human cells because important adipogenic transcription factors were up-regulated (PPARγ and C/EBPα) after miR-30c overexpression. Plasminogen activator inhibitor-1(PAI-1) and activin A receptor type I (ACVR1) are validated targets of this miRNA, which might explain its effect on adipogenesis [25]. Other pro-adipogenic miRNAs with a known target include miR-519, which regulates the transcription factor peroxisome proliferator activated receptor alpha (PPARA), which is involved in fatty acid oxidation, and miR-15a, which decreases cell proliferation when overexpressed and induces hypertrophy of murine adipocytes by fine-tuning of Delta-like 1 homolog levels (DLK1) [24]. Some miRNAs have an experimentally validated pro-adipogenic action, but their direct targets are still unknown, including miR-143 (the first miRNA found to be involved in mammalian adipogenesis), which is up-regulated during preadipocyte differentiation. The inhibition of miR-143 impairs differentiation, but when it is overexpressed by transfection, adipocytes accumulate more triglycerides in cell culture [24]. miR-103 is up-regulated during differentiation of murine adipocytes (3T3-L1), and its ectopic overexpression increases differentiation and lipid accumulation, with similar results being observed in porcine adipocytes. However, in human adipocytes miR-103 expression does not change significantly, remaining quite stable over a range of conditions. Therefore, the roles miR-103 plays in adipogenesis may not be the same among different mammalian species [24]. miR-378 is induced during adipogenesis of ST2 cells, when overexpressed, increases the size of triglyceride droplets in these cells. Its knockdown decreases lipid accumulation. miR-378 has also been associated with increased bovine backfat thickness [22]. miR-375 is likely to induce adipogenesis by modulating the ERK-PPARγ2-aP2 pathway. miR371 has been shown to increase adipocyte differentiation and might also be involved in epigenetic mechanisms controlling adipogenesis in vitro [26].

miRNAs That Inhibit Adipogenesis Adipogenesis is a complex process, and the differentiation stage is genetically controlled by transcriptional factors including PPARγ, which is fundamental for adipocyte differentiation, being considered the master adipogenesis regulator. PPARγ is the target of miR-27a/b which is down-regulated during the adipocyte differentiation of murine and human adipocytes cell lines. When overexpressed, miR-27 decreases PPARγ expression and reduces adipogenesis [27]. miR-130 also targets the 3′UTR, as well as the coding region, of PPARγ messenger RNA. Human primary preadipocyte differentiation in vitro is impaired when miR-130 is overexpressed, while it is increased when miR-130

549

550

CHAPTER 21:  Adipogenesis and Obesity

levels are reduced [22]. Like miR-17-92, let-7 impacts mitotic clonal expansion; however, it acts as an inhibitory regulator of this process. let-7 expression is down-regulated from day 0 to day 1 after hormonal i­ nduction in 3T3-L1 cell cultures. It is suggested that this temporal ­down-regulation allows mitotic clonal expansion; its expression then increases during adipocyte terminal differentiation. The ectopic expression of the pre-let-7a oligonucleotide inhibits mitotic clonal expansion and terminal differentiation. It is also suggested that let-7 inhibits adipogenesis by decreasing mRNA levels of the high-mobility group AT-hook 2 (HMGA2), a transcription factor that regulates proliferation and growth in other processes [22]. miR-369-5p targets the fatty acid–binding protein 4 (FABP4), which is a genetic marker of mature adipocytes. Overexpressed, this miRNA inhibits adipocyte differentiation and also seems to be involved in epigenetic adipogenesis control. Other anti-adipogenic miRNAs with known targets include miR-138, which targets EID1 (adenovirus early region 1-A-like inhibitor of differentiation 1) and miR-448, which targets Krüppel-like factor 5 (KLF5), a transcription factor that induces PPARγ expression. Overexpression of miR-138 or miR-448 in adipocyte cell cultures reduces differentiation [22]. miR-155, miR-221, and miR-222 were down-regulated in human mesenchymal stromal cells induced to differentiate into adipocytes. Their overexpression inhibited adipogenesis and repressed induction of the master regulators PPARγ and CCAAT/ enhancer-binding protein alpha. However, these miRNAs do not have experimentally validated targets [28]. The involvement of miRNAs in regulating adipogenesis might be greater, as only a few miRNA–mRNA interactions have been validated. The actual number of miRNAs that regulate each target has the potential to be larger according to bioinformatics miRNA binding site prediction. For example, miR-27a/b and miR-130 are known to directly target the PPARγ mRNA; however, a bioinformatics search (TargetScan) for potential miRNA binding sites in the 3′UTR of PPARγ shows 41 miRNA families with complimentary binding sequences (Figure 21.3). Some of these are true PPARγ regulators, while others may not be; this means that future studies are necessary to further validate miRNA– mRNA interactions, not only for PPARγ but for any gene of interest in the context of adipogenesis and obesity.

miRNA Changes during Adipocyte Differentiation miRNAs act in concert to regulate biological processes such as cellular differentiation. This is also true for adipocyte differentiation. Some miRNAs do not have their expression changed during differentiation because they may regulate basic cellular processes; however, others may have their expression levels altered (up- or down-regulated) to induce or inhibit more specialized

21.4  miRNA Regulation of Adipogenesis

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

Gene

Human PPARG NM_138712 3'UTR length:211

Conserved sites for miRNA families broadly conserved among vertebrates miR-130ac/301ab/301b/301b-3p/454/721/4295/3666 miR-27abc/27a-3p miR-128/128ab

Conserved sites for miRNA families conserved only among mammals Poorly conserved sites for miRNA families conserved among mammals or vertebrates

miR-340-5p

miR-24/24ab/24-3p

miR-590-3p

miR-34ac/34bc-5p/449abc/449c-5p miR-7/7ab

Sites for poorly conserved miRNA families miR-3664-3p

miR-3154

miR-4781-3p miR-409-3p miR-2441/4436a

miR-4446-5p

miR-4671-3p miR-891b miR-642a

miR-3185

miR-4755-5p

miR-513a-3p

miR-3662

miR-513a-5p miR-5096 miR-3140-3p

miR-3121-3p miR-548aaf

miR-3163

miR-4799-5p miR-1279

miR-4711-3p miR-511

miR-338-5p miR-586

miR-4284

miR-4775

miR-545

miR-548abakhjiwy/548abcd-5p/559 miR-548n/570

miR-4517

miR-548t/1643 miR-889

FIGURE 21.3  miRNA families predicted by TargetScan to target 3′UTR of PPARγ. miRNA families are aligned according to their predicted binding site location along the PPARγ 3′UTR. Source: from TargetScan search results.

cellular processes. Dynamic changes in miRNA expression lead to the inhibition or induction of genes that drive cellular differentiation. For example, adipocytes derived from subcutaneous adipose tissue in humans were isolated and induced to differentiate in vitro. A total of 70 miRNAs had altered expression (±1.2-fold, p < 0.0001) when preadipocytes (day 0) were compared to mature adipocytes after differentiation (day 14). The expression of 33 miRNAs increased during differentiation and the most up-regulated of these were miR378 (+6.6-fold), miR-30c (+5.1-fold), miR-30a (+4.0-fold), miR-30b (+3.1fold), miR-30e (+3.1-fold), miR-30a-3p (+2.8-fold), and miR-34a (+2.5 fold). Thirty-seven miRNAs had their expression reduced during adipocyte differentiation; the most down-regulated of these were miR-31-3p (−22.6-fold), miR210 (−24.3-fold), miR-221 (−24.9-fold), miR-424 (−24.6-fold), and miR-503 (−26.7-fold) [29]. Some of the up-regulated miRNAs (miR-378, miR-30a, and miR-30c) are known to have pro-adipogenic action. Similarly, miR-221 has an anti-adipogenic role and was down-regulated. The other miRNAs up- and down-regulated, however, do not have an established role in adipogenesis, which shows the need for further investigation to clarify the role of miRNAs most deregulated during human adipocyte differentiation. It is as yet unclear how miRNA expression is regulated in adipose tissue. In some miRNAs the DNA sequence is located between genes (i.e., the DNA’s intergenic regions) and are transcribed into RNA as independent units; in others the DNA sequence is located inside the host genes (e.g., in the introns). In this case, the miRNAs are transcribed simultaneously with the

551

552

CHAPTER 21:  Adipogenesis and Obesity

genes that host their sequences. Transcription factors are known to regulate the expression of genes, and may also participate in miRNA regulation by promoting the expression of miRNA genes or the transcription of miRNA host genes. In adipogenesis, PPARγ is the most influential transcription factor because it has thousands of genomic binding sites, indicating its important regulatory role. A study attempted to find out whether miRNA expression is under the regulation of PPARγ and identified 22 putative PPARγ target miRNA genes with PPARγ binding sites within ±50 kilobases distance from their transcription start site [30]. Some of these miRNAs were located inside protein-coding genes, while others had their own genes. Among these, the study further verified that the expression of miR-103-1 (inside gene PANK3), miR-148b (inside gene COPZ1), miR-182/96/183 (own miRNA gene), miR-205 (own miRNA gene), and miR-378 (inside gene PPARGC1B) followed that of PPARγ during in vitro adipocyte differentiation [30]. These findings shed some light on how miRNAs might be regulated in adipose tissue and improve our understanding on how the regulatory network of miRNAs and transcription factors control gene expression of adipocytes (see Table 21.1).

21.5  miRNAs IN OBESITY miRNAs play a role in obesity; several are up-regulated or down-regulated when their expression is compared between lean and obese individuals (Figure 21.4). Changes in miRNA expression in obesity alter the regulation of genes controlling adipose tissue development and metabolism, leading to metabolic dysfunctions. More interestingly, some miRNAs normally up-regulated during normal adipocyte differentiation were down-regulated in adipose tissue of obese individuals, while others were down-regulated during differentiation but up-regulated in obesity. These inverted patterns of miRNA expression suggest the involvement of miRNAs in obesity’s dysfunctional adipocyte regulation. Obesity affects miRNA expression not only in adipose tissue but also in other organs such as pancreas and liver, which are also reported to have abnormal miRNA expression in obese individuals [24]. miRNAs differentially expressed in mice and human obesity models are discussed in the following sections.

SECTION KEY POINT miRNA expression is altered in obese individuals, and many miRNAs have an inverse expression

pattern compared to normal adipogenesis, indicating a dysfunctional adipogenesis in obesity.

21.5  miRNAs in Obesity

FIGURE 21.4  miRNAs up- and down-regulated in human and mouse obesity models.

21.5.1 Mouse Studies As mentioned earlier, several miRNAs have an inverse regulatory pattern during adipogenesis and obesity. For example, one study found expression of miR-422b, miR-148a, miR-103, miR-107, miR-30c, miR-30a-5p, and miR-143 to be induced during murine cell (3T3-L1) adipogenesis, but down-regulated in adipose cells from a diet-induced obesity mouse model. On the other hand, miR-221 and miR-222 were found to be down-regulated during adipogenesis in vitro and up-regulated in obese conditions [31]. It has been hypothesized that the inverted miRNA expression pattern between adipogenesis and obesity is associated with the obesity-caused chronic inflammatory environment of adipose tissue. Adipocyte hypertrophy and hyperplasia in obesity leads to inadequate blood circulation, which causes hypoxia; hypoxia can be one of the triggers of inflammation by the infiltration of macrophages, which secrete TNF-α, a proinflammatory adipokine. This hypothesis was tested by treating differentiated adipocytes in vitro with TNF-α to mimic chronic inflammation. The result was that most miRNAs had similar expression patterns between obese adipose tissue and adipocytes treated with TNF-α, supporting the idea that adipose tissue inflammation promotes abnormal miRNA expression [31].

553

554

CHAPTER 21:  Adipogenesis and Obesity

miR-27a and miR-24b are down-regulated during adipogenesis and inhibit this process. In one study, obese mice (ob/ob) showed up-regulated levels of ­miR-27a/b in epididymal fat, and it was further determined that miR-27a/b expression is responsive to the hypoxia in vitro that is also expected in obesity. These results indicate that miR-27 might play a role in obesity pathophysiology [24]. Although some miRNAs present an inverse expression pattern between adipogenesis and obesity models, others do not follow this trend. For instance, m ­ iR-335 was up-regulated in WAT and liver in three different mice obesity models (KKAy44 mice, leptin-deficient ob/ob mice, and leptin receptor–deficient db/db mice). It was also highly induced during adipogenesis of 3T3-L1 cells, being well correlated with the expression of adipocyte differentiation markers—fatty acid synthase (FASN), FABP4, and PPARγ. The results indicated that the ­up-regulation of miR335 in adipose tissue and liver plays a role in the pathophysiology of ­obesity; however, no direct target for this miRNA has been found so far [27]. Another high-throughput experiment detected 22 miRNAs differentially expressed between wild-type mice fed a standard diet and a group fed a long-term ­obesity-inducing diet. A total of 8 miRNAs were up-regulated (miR-21, miR-142-3p, miR-142-5p, miR-146a, miR-146b, miR-222, miR-342-3p, and miR-379), while 14 were down-regulated (miR-1, miR-30a-3p, miR-30e-3p, miR-122, miR-130a, miR-133b, miR-192, miR-193a-3p, miR-200b, miR-200c, miR-203, miR-204, and miR-378) [32]. Some of these miRNAs are already known to impact adipogenesis; however, further studies are necessary to determine the roles of the others.

21.5.2 Human Studies Human obesity disrupts the physiological regulation of adipose tissue, and abnormal miRNAs levels are part of this condition. For example, ­preadipocytes from the adipose tissue of obese or lean individuals have different miRNA expression profiles when induced to differentiate into mature adipocytes. In one study, the expression of 40 miRNAs was different between preadipocytes derived from lean individuals and those derived from obese individuals, while 31 were differentially expressed after these preadipocytes were differentiated. A total of 21 differentially expressed miRNAs were common in preadipocytes and adipocytes. In the same study, miRNA expression in vivo from the subcutaneous fat of obese versus lean individuals showed that the expression of 9 miRNAs was different with 4 miRNAs (miR-130b, miR-139-5p, miR-185, and miR-484) down-regulated and 5 miRNAs (miR-99a, miR-125b, miR-199a-5p, miR-221, and miR-1229) u ­ p-regulated in obese individuals. In addition, 17 miRNAs presented significant correlations (<0.05) with BMI and/or metabolic parameters [29]. Another study investigating miRNAs differentially expressed between lean and obese individuals used microarray analysis to detect two miRNAs ­down-regulated in the omental fat of obese individuals. These were miR-17-5p

21.6  Factors That Influence miRNA Expression in Adipose Tissue

and miR-132. Interestingly, the levels of these two miRNAs both in blood and fat tissue correlated well with fasting blood glucose, BMI, and glycosylated hemoglobin, which makes them potential miRNA biomarkers for obesity [33]. Other studies focused on specific miRNAs in obesity instead of large miRNA profiles. miR-519d was up-regulated in the subcutaneous adipose t­issue of the severely obese (BMI = 42.7 ± 1.2) when compared to the nonobese (BMI = 24.7±1.6). The high level of miR-519d in vivo was accompanied by a low protein level of PPARα, a transcriptional factor involved in fatty acid oxidation. To evaluate miR-519d effects in vitro, miR-519d was overexpressed during adipocyte differentiation, leading to a dose-dependent reduction in PPARα translation and to increased lipid accumulation. These results suggested that the high expression of miR-519d in obesity disrupts fatty acid homeostasis and promotes adipocyte hypertrophy [34]. miRNAs might be important regulators of inflammation in adipose tissue of obese humans. Subcutaneous adipose tissue of obese individuals produces high amounts of proinflammatory proteins, including chemokine (C‒C motif) ligand 2 (CCL2), a protein that has been suggested to start the inflammation process by attracting the inflammatory cells from circulation. A study of miRNAs profiles between the subcutaneous fat of obese and lean women (adipocyte fraction only) found 11 miRNAs (miR-26a, miR-30c, m ­ iR-92a, miR-126, m ­ iR-143, miR-145, miR-193a, miR-193b, miR-652, let-7a, and let-7d) down-regulated in obese group. When overexpressed, two of these miRNAs (miR-126 and miR-193b) repressed CCL2 expression in cell culture. ­miR-126 directly targeted CCL2 messenger RNA, while miR-193b regulated CCL2 expression indirectly through a network of transcription factors. This shows the role of miRNAs in the inflammation of obese adipose tissue [35]. It is important to consider that the results from miRNA-profiling studies vary considerably according to some experimental characteristics, including species, cell culture model, and fat depot used. There is also the type of tissue sample tested, as some studies use whole adipose tissue, which includes several cell types (adipocytes, preadipocytes, macrophages, etc.); others isolate only the adipocytes from the sample. These factors among others should be taken into account when comparing results from different studies analyzing miRNAs in adipogenesis and obesity.

21.6 FACTORS THAT INFLUENCE miRNA EXPRESSION IN ADIPOSE TISSUE miRNA expression in adipose tissue changes according to a variety of conditions. Dietary manipulation has been shown to change the expression of protein-coding genes; the same is observed with miRNAs. Even early-life

555

556

CHAPTER 21:  Adipogenesis and Obesity

dietary changes might induce long-term miRNA expression changes. Brown and white adipose tissues express different miRNA profiles and changes are also observed among different anatomical locations of fat depots: visceral, subcutaneous, and intramuscular. These factors, among others (Figure 21.5), should be taken into account in order to fully understand miRNA regulation in adipose tissue. The influence of dietary changes and adipose depot type are discussed next.

SECTION KEY miRNA expression is affected by external factors,

such as diet, and internal factors, such as different depot types and adipose tissue locations.

FIGURE 21.5  Factors that affect miRNA expression in adipose tissue.

21.6.1 Diet The expression of protein-coding genes in adipose tissue is already known to be influenced by dietary manipulation; however, the nature of this influence is relatively unknown. A high-fat diet is the most common strategy for investigating the effects of diet on miRNA expression, as it provides diet-induced obesity models. For example, mice fed a high-fat diet for 8 weeks had increased miR-143 expression in mesenteric adipose tissue; miR-143 levels were highly correlated to the expression of PPARγ, FABP4, and plasma leptin levels [27]. miR-27a was down-regulated in the mature adipocyte fraction of adipose tissue

21.6  Factors That Influence miRNA Expression in Adipose Tissue

of mice fed a high-fat diet [27]. In one study, miRNA expression was found to be influenced by a 10% flax seed supplement in the bovine diet for 14 weeks, which elevated total dietary fat from 1.95% to 5.85%. Eight out of twelve selected miRNAs (miR-19a, miR-92a, miR-92b, miR-101, miR-103, miR-106, miR-142-5p, and miR-296) were confirmed to be significantly up-regulated in response to a flax diet [36]. Conjugated linoleic acid (CLA) is proposed to promote weight loss, and a study reported that CLA supplementation in a mouse diet reduced the expression of miR-143 and miR-107 in retroperitoneal adipose tissue, while it increased the expression of miR-222 [37]. These findings reveal that miRNAs expression responds to dietary changes. Even early-life nutrition seems to play a role in miRNA expression. In one study, female rats were fed a low-protein diet during pregnancy and lactation, which led to reduced adipose mass and adipocyte size in offspring. Microarray analyses showed that miR-483-3p was the only miRNA up-regulated at offspring age 22 days and 3 months, indicating that this change was a sustained programmed event. The overexpression of miR-483-3p in 3T3-L1 cell culture inhibited adipogenesis by targeting growth differentiation factor 3 (GDF3) directly, which is required for lipid accumulation during late stages of adipocyte differentiation. The exact mechanism controlling miR-483-3p programming is still unknown. Interestingly, in humans from a similar model of suboptimal early nutrition (those with low birth weight), miR-483-3p expression was up-regulated with repressed expression of GDF3, suggesting that this is a conserved mechanism with potential long-term health consequences [38].

21.6.2 Adipose Depots As mentioned earlier, the adipose organ is composed of two tissue types, WAT and BAT. These distinct tissues differ morphologically and functionally. BAT is present in mammals, being well known in rodents and human neonates [3]. In adult humans the function of brown adipocytes was believed to be insignificant, but recent findings have shown that BAT is present and can be activated under cold exposure. More importantly, BAT was more prevalent in lean as compared to obese individuals, which makes it an interesting target in obesity treatment [39]. Understanding the differences in miRNA expression regulation between BAT and WAT might be an answer in obesity control. In theory, converting WAT into BAT would promote the expenditure of fat reserves into heat, reducing adipose mass and consequently obesity. Evidence shows that miRNA expression in brown adipocytes differs from that in white adipocytes [40]. For example, miR-143 was low-expressed in mature brown adipocytes of mice but high-expressed in white adipocytes. miR-455 was up-regulated during brown adipocyte differentiation and was

557

558

CHAPTER 21:  Adipogenesis and Obesity

speculated to have a pro-adipogenic role. Interestingly three miRNAs normally expressed in myocytes (miR-1, miR-133a, and miR-206) were not detected in white adipocytes but were expressed in brown adipocytes. One study identified miR-196a as a potential controller of the conversion of WAT into BAT [41]. miR-196a is up-regulated during brown adipocyte differentiation and targets the white fat gene homeobox C8 (HOXC8). HOXC8 is down-regulated during brown fat adipogenesis and represses the expression of brown fat genes, including the master regulator of brown adipogenesis. To test miR-196a in vivo, transgenic miR-196a mice were developed to promote forced expression of miR-196a in adipose tissue. The results showed that this miRNA increased the amount of BAT in adipose tissue, increased energy expenditure and promoted obesity resistance [41]. These findings open the possibilities of exploring miRNAs as therapeutic tools for controlling obesity by taking advantage of the differential molecular regulation of BAT and WAT [41]. miRNA expression is different not only between WAT and BAT but also among different locations of WAT, such as visceral, intramuscular, and subcutaneous adipose tissues. A high-throughput study profiling 1146 human miRNAs found in abdominal and subcutaneous gluteus fat from 70 individuals revealed that 136 miRNAs (12%) were differentially expressed between the two locations, with 61 having higher expression in gluteal fat and 75 having higher expression in abdominal fat [42]. Distinct miRNA regulation in different fat depots is further supported by a study that found the correlation between the expression of 95 miRNAs from the subcutaneous and omental adipose tissue in 50 individuals to be insignificant (r = −0.187, p = 0.07) [33]. In another study comparing miRNA expression between omental and subcutaneous adipose tissue from individuals with normal glucose tolerance and from individuals with type 2 diabetes (T2D), it was reported that in the first group 4 miRNAs were differentially expressed between fat depots, while in the second group 12 miRNAs were differentially expressed [43]. Similar depot-specific miRNA regulation has been reported in bovine adipose depots (subcutaneous versus visceral fat) [36], and different miRNA profiles have been reported even in three different locations at the same (subcutaneous fat) depot [22]. This supports the effect of tissue type on miRNA expression. A porcine study using primary adipocytes found 30 miRNAs expressed in a depot-specific manner, of which 24 were expressed only in intramuscular fat and 6 were expressed only in subcutaneous fat [44]. All of these findings from different mammalian species support the notion that different adipose tissues have different miRNA regulation, reflecting their diverse physiology.

21.7  Conclusions and Future Perspectives

21.7 CONCLUSIONS AND FUTURE PERSPECTIVES Obesity is now a worldwide epidemic. Efforts are being made to further understand the molecular regulation of adipose tissue as a way of developing therapeutic strategies to treat this condition. Because miRNAs regulate the expression of adipose tissue genes, a number of studies have attempted to profile their expression during adipogenesis using in vitro models as well as in vivo models using the adipose tissue of both lean and obese individuals. Our understanding of the roles of miRNAs in obesity is increasing as studies continue to show that several miRNAs in lean and obese subjects are differentially expressed. Several miRNAs have been identified as increasing adipocyte differentiation, while others have been shown to play an anti-adipogenic role. The regulatory activity of these miRNAs affects genes involved in several steps of adipogenesis, including commitment, clonal expansion, and differentiation (Figure 21.6). However, because many pro- and anti-adipogenic miRNAs do not have a well-defined regulatory

FIGURE 21.6  miRNAs involved in adipogenesis miRNAs in the dark green boxes favor adipogenesis; those in dark red boxes inhibit it. Proteins or pathways in light green favor adipogenesis; those in light red inhibit it. Lines with an arrow end indicate induction effect, while those with T end indicate inhibition.

559

560

CHAPTER 21:  Adipogenesis and Obesity

mechanism in adipogenesis, further investigation must be carried out to determine their direct targets. Evidence further indicates that adipocyte differentiation in obesity is abnormal. This evidence is supported by the fact that many miRNAs have an inverse expression pattern between obese adipose tissue and normal adipogenesis in cell culture. Understanding how miRNAs regulate gene expression in adipose tissue requires taking into consideration that miRNA profiles may change because of external and internal environmental factors. For example, diet can significantly alter miRNA expression profiles. These profiles are also distinct according to type of adipose tissue (WAT or BAT) and the different locations of fat depots (e.g., visceral, subcutaneous, and intramuscular). These differences reflect the different physiology of each depot and show that miRNAs have a dynamic role in adipose physiology that can change depending on several conditions. miRNAs are important regulators of adipose tissue development and functions. Moreover, abnormal miRNA levels have been reported in obesity, which makes the development of miRNA-based therapies a promising tool in obesity treatment. Our understanding of miRNA regulation of adipogenesis has grown considerably with the finding from different studies that several miRNAs enhance or inhibit adipogenesis [22]. A great part of our knowledge of the roles of miRNAs in adipocytes is derived from cell cultures, which are excellent controlled models for studying adipogenesis. However, these studies could not fully explain what happens in vivo. The complexity of the biological processes involved in adipogenesis and obesity development in vivo is much greater than that in isolated cell systems, and therefore more studies using in vivo models are necessary to fully translate the biological roles of miRNAs in fat development and obesity. It has become clear that miRNAs are important in adipogenesis and obesity pathogenesis, which is why there is great interest in harvesting their regulatory power as a new therapeutic tool in obesity treatment [45]. Manipulating miRNA levels in vivo as a way to treat adipose dysfunction might require increasing the expression of a specific miRNA through synthetic miRNA mimetics or repressing it with the antisense oligonucleotides. It is known that miRNAs control gene expression by complementary binding to the 3′UTR of messenger RNAs. Under many conditions, including obesity, miRNA expression can be significantly altered [23], meaning that the genes regulated by these miRNAs are consequently likely to be dysregulated as well. In this context, the development of miRNA therapies to restore normal levels of altered miRNAs might be one of the best options for restoring the expression of several genes targeted by them.

Chapter Questions

Several complications make miRNAs as therapeutic targets a challenge. In obesity, for example, not just one miRNA might be altered; in fact, as described previously, several miRNAs can be up- or down-regulated. Besides, each miRNA has the potential to regulate many genes, which means that miRNA therapy might lead to undesirable off-target effects. Another concern is the need to deliver therapeutic nucleic acids specifically to the organs of interest. Adipose tissue might be specially challenging as it not a centralized organ with different fat depots and so methods will have to be developed to deliver miRNAs or miRNA inhibitors in a tissue-specific manner. However, the potential of miRNA therapy directed at biological pathways in obesity is very promising. miRNAs might also be used as biomarkers of adipose tissue function, especially those whose levels in peripheral circulation are correlated with adipose tissue, because sampling blood is less invasive and more viable than sampling adipose tissue. Some miRNAs show promise in filling these requirements, such miR-17-5p and miR-132, which are differentially expressed between obese and lean individuals and correlate significantly with BMI, fasting blood glucose, and glycosylated hemoglobin [33].

CHAPTER QUESTIONS Short-Answer 1. Briefly define adipogenesis and its two main stages: commitment and differentiation. Answer: Adipogenesis is the process by which nonspecialized stem cells are converted into mature adipocytes. This process can be divided in two main stages: commitment and differentiation. Commitment involves the conversion of multipotent stem cells into preadipocytes that are solely committed to the adipose lineage. Differentiation follows and converts the preadipocytes into mature adipocytes, which are capable of secreting adipokines and transporting, synthesizing, and releasing lipids. 2. Briefly describe how miRNAs can impact adipogenesis. Answer: miRNAs affect adipogenesis at the commitment and differentiation stages. During commitment, they may favor the adipose lineage or direct the multipotent stem cells to other cell types. During the differentiation, pro-adipogenic miRNAs may repress the expression of genes that inhibit adipogenesis, therefore enhancing the process; anti-adipogenic miRNAs may target genes that favor adipogenesis and, in this case, inhibit adipogenesis.

561

562

CHAPTER 21:  Adipogenesis and Obesity

3. How is miRNA expression in adipose tissue altered by obesity? Answer: Several miRNAs present abnormal expression (up- or down-regulation) in obese adipose tissue when compared to lean tissue. Obesity leads to a dysfunctional regulation of adipocyte differentiation; this is supported by the fact that many miRNAs in obesity show an inverse pattern of expression when compared to normal differentiation of adipocytes in vitro. Evidence shows that inflammation in obese tissue is one of the triggers of abnormal miRNA expression in obesity. 4. What factors must be taken into account when analyzing results from miRNA studies of adipose tissue or adipocytes in cell culture systems? Answer: Several factors can affect how miRNAs are expressed. In cell culture systems, cell line type is an important variable. The behavior of miRNAs in vivo can be affected by species of interest, adipose tissue (WAT or BAT), location of fat depots (visceral, subcutaneous, or intramuscular), gender, adiposity status (lean or obese), and environmental factors such as diet. 5. How can miRNAs be used as therapeutic tools in the treatment of obesity? Answer: miRNA expression is altered in many conditions, including obesity. Abnormal expression participates in obesity pathophysiology by modifying the normal expression levels of target genes. In this context, miRNA therapies can be based on the restoration of normal levels of altered miRNAs in order to correct gene expression. This is achieved by increasing the expression of a down-regulated miRNA through synthetic miRNA mimetics or inhibition of the action of an abnormally up-regulated miRNA through the use of antisense oligonucleotides.

REFERENCES [1] Poulos SP, Hausman DB, Hausman GJ. The development and endocrine functions of adipose tissue. Mol Cell Endocrinol 2010;323:20–34. [2] Large V, Peroni O, Letexier D, Ray H, Beylot M. Metabolism of lipids in human white adipocyte. Diab Metab 2004;30:294–309. [3] Hansen JB, Kristiansen K. Regulatory circuits controlling white versus brown adipocyte differentiation. Biochem J 2006;398:153–68. [4] Wang S, Soni KG, Semache M, Casavant S, Fortier M, et al. Lipolysis and the integrated physiology of lipid energy metabolism. Mol Genet Metab 2008;95:117–26. [5] Rosen ED, Spiegelman BM. Adipocytes as regulators of energy balance and glucose homeostasis. Nature 2006;444:847–53. [6] Galic S, Oakhill JS, Steinberg GR. Adipose tissue as an endocrine organ. Mol Cell Endocrinol 2010;316:129–39.

References

[7] Lago F, Gómez R, Gómez-Reino JJ, Dieguez C, Gualillo O. Adipokines as novel modulators of lipid metabolism. Trends Biochem Sci 2009;34:500–10. [8] Filková M, Haluzík M, Gay S, Senolt L. The role of resistin as a regulator of inflammation: Implications for various human pathologies. Clin Immunol 2009;133:157–70. [9] Moreno-Navarrete JM, Fernández-Real JM. Adipocyte Differentiation. In: Symonds ME, editor. Adipose Tissue Biology. Springer/Dordrecht Heidelberg; 2012. pp. 17–38. [10] Huang H, Song TJ, Li X, Hu L, He Q, et al. BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage. Proc Nat Acad Sci USA 2009;106:12670–5. [11] McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 2004;6:483–95. [12] Gregoire FM, Smas CM, Sul HS. Understanding adipocyte differentiation. Physiol Rev 1998;78:783–809. [13] Lefterova MI, Lazar MA. New developments in adipogenesis. Trends Endocrinol Metab 2009;20:107–14. [14] Nguyen T, Lau DCW. The obesity epidemic and its impact on hypertension. Can J Cardiol 2012;28:326–33. [15] Bray GA. Epidemiology, risks and pathogenesis of obesity. Meat Sci 2005;71:2–7. [16] Wilding JPH. Pathophysiology and aetiology of obesity. Medicine 2006;34:501–5. [17] Guh D, Zhang W, Bansback N, Amarsi Z, Birmingham CL, et al. The incidence of co-morbidities related to obesity and overweight: A systematic review and meta-analysis. BMC Public Health 2009;9:88. [18] Brown WV, Fujioka K, Wilson PWF, Woodworth KA. Obesity: Why be concerned? Am J Med 2009;122:I–CO4. [19] Haslam DW, James WPT. Obesity. Lancet 2005;366:1197–209. [20] Shoelson SE, Herrero L, Naaz A. Obesity, Inflammation, and Insulin Resistance. Gastroenterology 2007;132:2169–80. [21] Friedman RC, Farh KK-H, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 2009;19:92–105. [22] Romao JM, Jin W, Dodson MV, Hausman GJ, Moore SS, et al. MicroRNA regulation in mammalian adipogenesis. Exp Biol Med 2011;236:997–1004. [23] Alexander R, Lodish H, Sun L. MicroRNAs in adipogenesis and as therapeutic targets for obesity. Expert Opin Ther Targets 2011;15:623–36. [24] Hilton C, Neville MJ, Karpe F. MicroRNAs in adipose tissue: their role in adipogenesis and obesity. Int J Obes 2012. 2012/04/24/online: 1–8. [25] Karbiener M, Neuhold C, Opriessnig P, Prokesch A, Bogner-Strauss JG, et al. MicroRNA-30c promotes human adipocyte differentiation and co-represses PAI-1 and ALK2. RNA Biol 2011;8:850–60. [26] Bork S, Horn P, Castoldi M, Hellwig I, Ho AD, et al. Adipogenic differentiation of human mesenchymal stromal cells is down-regulated by microRNA-369-5p and up-regulated by microRNA-371. J Cell Physiol 2011;226:2226–34. [27] A. McGregor R,S, Choi M. microRNAs in the regulation of adipogenesis and obesity. Curr Mol Med 2011;11:304–16. [28] Skårn M, Namløs HM, Noordhuis P, Wang M-Y, Meza-Zepeda LA, et al. Adipocyte differentiation of human bone marrow-derived stromal cells is modulated by microRNA-155, microRNA-221, and microRNA-222. Stem Cells Dev 2012;21:873–83.

563

564

CHAPTER 21:  Adipogenesis and Obesity

[29] Ortega FJ, Moreno-Navarrete JM, Pardo G, Sabater M, Hummel M, et al. miRNA expression profile of human subcutaneous adipose and during adipocyte differentiation. PLoS One 2010;5:e9022. [30] John E, Wienecke-Baldacchino A, Liivrand M, Heinäniemi M, Carlberg C, et al. Dataset integration identifies transcriptional regulation of microRNA genes by PPARγ in differentiating mouse 3T3-L1 adipocytes. Nucleic Acids Res 2012;40(10):4446–60. [31] Xie H, Lim B, Lodish HF. MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity. Diabetes 2009;58:1050–7. [32] Chartoumpekis DV, Zaravinos A, Ziros PG, Iskrenova RP, Psyrogiannis AI, et al. Differential expression of microRNAs in adipose tissue after long-term high-fat diet-induced obesity in mice. PLoS One 2012;7:e34872. [33] Heneghan HM, Miller N, McAnena OJ, O’Brien T, Kerin MJ. Differential miRNA expression in omental adipose tissue and in the circulation of obese patients identifies novel metabolic biomarkers. J Clin Endocrinol Metab 2011;96:E846–50. [34] Martinelli R, Nardelli C, Pilone V, Buonomo T, Liguori R, et al. miR-519d Overexpression is associated with human obesity. obesity 2010;18:2170–6. [35] Arner E, Mejhert N, Kulyté A, Balwierz PJ, Pachkov M, et al. Adipose tissue microRNAs as regulators of CCL2 production in human obesity. Diabetes 2012;61:1986–93. [36] Romao JM, Jin W, He M, McAllister T, Guan LL. Altered microRNA expression in Bovine subcutaneous and visceral adipose tissues from Cattle under different diet. PLoS One 2012;7:e40605. [37] Parra P, Serra F, Palou A. Expression of adipose microRNAs is sensitive to dietary conjugated linoleic acid treatment in mice. PLoS One 2010;5:e13005. [38] Ferland-McCollough D, Fernandez-Twinn DS, Cannell IG, David H, Warner M, et al. Programming of adipose tissue miR-483-3p and GDF-3 expression by maternal diet in type 2 diabetes. Cell Death Differ 2012;19:1003–12. [39] van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, Drossaerts JMAFL, Kemerink GJ, et al. Cold-activated brown adipose tissue in healthy men. New Engl J Med 2009;360:1500–8. [40] Walden TB, Timmons JA, Keller P, Nedergaard J, Cannon B. Distinct expression of muscle-specific microRNAs (myomirs) in brown adipocytes. J Cell Physiol 2009;218:444–9. [41] Mori M, Nakagami H, Rodriguez-Araujo G, Nimura K, Kaneda Y. Essential Role for miR-196a in Brown adipogenesis of white fat progenitor cells. PLoS Biol 2012;10:e1001314. [42] Rantalainen M, Herrera BM, Nicholson G, Bowden R, Wills QF, et al. MicroRNA expression in abdominal and gluteal adipose tissue is associated with mRNA expression levels and partly genetically driven. PLoS One 2011;6:e27338. [43] Klöting N, Berthold S, Kovacs P, Schön MR, Fasshauer M, et al. MicroRNA expression in human omental and subcutaneous adipose tissue. PLoS One 2009;4:e4699. [44] Guo Y, Mo D, Zhang Y, Zhang Y, Cong P, et al. MicroRNAome comparison between intramuscular and subcutaneous vascular stem cell adipogenesis. PLoS One 2012;7:e45410. [45] Xie H, Sun L, Lodish HF. Targeting microRNAs in obesity. Expert Opin Ther Targets 2009;13:1227–38.

ONLINE RESOURCES www.iaso.org/. The International Association for the Study of Obesity (IASO). www.nature.com/ ijo/index.html. The International Journal of Obesity (IJO). www.mirbase.org/. The miRBase database. onlinelibrary.wiley.com/journal/10.1111/(ISSN)1467–789X. Obesity Reviews.

Further Reading

FURTHER READING Gimble JM, Bunnell BA. Adipose-Derived Stem Cells. Dordrecht Heidelberg Springer; 2011. Fantuzzi G, Mazzone T. Adipose Tissue And Adipokines in Health And Disease. Humana Press; 2007. Symonds ME, editor. Adipose Tissue Biology. Dordrecht, Heidelberg: Springer; 2012. Yang K, editor. Adipose Tissue Protocols. Humana Press; 2008.

565