Download Introduction to Adipose Tissue Adipose tissue is not merely an

Introduction to Adipose Tissue
Adipose tissue is not merely an organ designed to passively store excess carbon in the form of fatty acids
esterified to glycerol (triglycerides). Mature adipocytes synthesize and secrete numerous enzymes,
growth factors, cytokines and hormones that are involved in overall energy homeostasis. Many of the
factors that influence adipogenesis are also involved in diverse processes in the body including lipid
homeostasis and modulation of inflammatory responses. In addition, a number of proteins secreted by
adipocytes play important roles in these same processes. In fact recent evidence has demonstrated that
many factors secreted from adipocytes are pro-inflammatory mediators and these proteins have been
termed adipocytokines or adipokines. There are currently over 50 different adipokines recognized as
being secreted from adipose tissue. These adipokines are implicated in the modulation of a range of
physiological responses that globally includes appetite control and energy balance. Specific metabolic
processes regulated by adipose tissue include lipid metabolism, glucose homeostasis, inflammation,
angiogenesis, hemostasis (regulation of blood coagulation), and blood pressure.
The major form of adipose tissue in mammals (commonly referred to as "fat") is white adipose tissue,
WAT. Specialized adipose tissue that is primarily tasked with thermogenesis, especially in the neonate, is
brown adipose tissue, BAT. BAT is so-called because it is darkly pigmented due to the high density of
mitochondria rich in cytochromes. BAT specializes in the production of heat (adaptive thermogenesis)
and lipid oxidation.
Histological section of adipose tissue demonstrating distinctive morphology of WAT and BAT. White
adipocytes occupy the left side of the image and brown adipocytes the right side. As described below
white adipocytes are generally rounded with over 90% of the cell volume taken up by a single fat
droplet. The few small mitochondria and the nucleus are compressed to the very edge of the white
adipocyte. The brown adipocytes are smaller in overall size, polygonal in shape, contain several small
lipid droplets and high numbers of large mitochondria which imparts the brown color to these cells.
Image from Junqueira’s Basic Histology, 12th ed. by Anthony L. Mescher, McGraw-Hill Professional
Division, reproduced with permission.
WAT is composed of adipocytes held together by a loose connective tissue that is highly vascularized
and innervated. White adipocytes are rounded cells that contain a single large fat droplet that occupies
over 90% of the cell volume. The mitochondria within white adipocytes are small and few in number.
The mitochondria and nucleus of the white adipocyte is squeezed into the remaining cell volume.
Molecular characteristics of white adipocytes include expression of leptin but no expression of
uncoupling protein 1, UCP1 (designated UCP1–, leptin+). Brown adipocytes are smaller in overall size
compared to white adipocytes. Brown adipocytes are polygonal in shape and contain numerous large
mitochondria packed with cristae. Whereas, white adipocytes contain a single large fat droplet, brown
adipocytes contain several small lipid droplets. Brown adipocytes are molecularly UCP1+ and leptin–. BAT
is primarily visceral with highest concentrations around the aorta. BAT is highly vascularized and
contains a very high density of noradrenergic nerve fibers.
In addition to adipocytes, WAT contains macrophages, leukocytes, fibroblasts, adipocyte progenitor
cells, and endothelial cells. The presence of the fibroblasts, macrophages, and other leukocytes along
with adipocytes, accounts for the vast array of proteins that are secreted from WAT under varying
conditions. The highest accumulations of WAT are found in the subcutaneous regions of the body and
surrounding the viscera (internal organs of the chest and abdomen).
Although WAT can be found associated with numerous organs its functions are more than just insulation
of the organ and a ready reservoir of fat for energy production. Depending on its location WAT serves
specialized functions. The WAT associated with abdominal and thoracic organs (excluding the heart), the
so-called visceral fat, secretes several inflammatory cytokines and is thus involved in local and systemic
inflammatory processes. WAT associated with skeletal muscle secretes free fatty acids, interleukin-6 (IL6) and tumor necrosis factor-α (TNFα) and as a consequence plays a significant role in the development
of insulin resistance. Cardiac tissue associated WAT secretes numerous cytokines resulting in local
inflammatory events and chemotaxis that can result in the development of atherosclerosis and systolic
hypertension. Kidney associated WAT plays a role in sodium reabsorption and therefore can affect
intravascular volume and hypertension.
The major focus of this discussion will be on the biological activities associated with WAT, however,
discussion of BAT is included at the end of this page. WAT serves many functions including insulating the
viscera, storing excess carbon energy in the form of triglycerides and mediating glucose homeostasis.
WAT also plays important roles as an endocrine/immune organ by secreting adipokines that includes
inflammatory cytokines, complement-like factors, chemokines, and acute phase proteins. The endocrine
functions of WAT regulate appetite, energy metabolism, glucose and lipid metabolism, inflammatory
processes, angiogenesis, and reproductive functions.
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Regulation of Adipogenesis
The process of adipocyte differentiation from a precursor preadipocyte to a fully mature adipocyte
follows a precisely ordered and temporally regulated series of events. Adipocyte precursor cells emerge
from mesenchymal stem cells (MSCs) that are themselves derived from the mesodermal layer of the
embryo. The pluripotent MSCs receive extracellular cues that lead to the commitment to the
preadipocyte lineage. Preadipocytes cannot be morphologically distinguished from their precursor MSCs
but they have lost the ability to differentiate into other cell types. This initial step in adipocyte
differentiation is referred to as determination and leads to proliferating preadipocytes undergoing a
growth arrest. This initial growth arrest occurs coincident with the expression of two key transcription
factors, CCAAT/enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor
gamma (PPARγ). Following the induction of these two critical transcription factors there is a permanent
period of growth arrest followed by expression of the fully differentiated adipocyte phenotype. This
latter phase of adipogenesis is referred to as terminal differentiation.
Although PPARγ and C/EBPα are the most important factors regulating adipogenesis additional
transcription factors are known to influence this process. These additional factors include sterolregulated element binding protein 1c (SREBP1c, also known as ADD1 for adipocyte differentiation-1),
signal transducers and activators of transcription 5 (STAT5), AP-1 and members of the Krüppel-like factor
(KLF4, KLF5, and KLF15) family as well as C/EBP beta (β) and C/EBP delta (δ). More information about the
roles of PPARγ and SREBP in metabolic homeostasis can be found in the PPAR page as well as the
Cholesterol page. Although these numerous transcription factors have been shown to influence overall
adipogenesis, either positively or negatively, PPARγ is the only one that is necessary for adipogenesis to
take place. In fact, in the absence of PPARγ, adipocyte differentiation fails to occur and as yet no factor
has been identified that can rescue adipogenesis in the absence of PPARγ. In spite of this fact,
expression of PPARγ does not commence during the initial activation of adipocyte differentiation but
only after the responses elicited by STAT5, KLF4, KLF5, AP-1, SREBP1c, and C/EBPβ and C/EBPδ are
exerted.
PPARγ was originally identified as being expressed in differentiating adipocytes and as indicated above it
is now recognized as a master regulator of adipogenesis. PPARγ was identified as the target of the
thiazolidinedione (TZD) class of insulin-sensitizing drugs. The mechanism of action of the TZDs is a
function of the activation of PPARγ and the consequent induction of genes necessary for differentiation
of adipocytes. The human PPARγ gene (symbol PPARG) is located on chromosome 3p25 spanning over
100kb and composed of 9 exons encoding two biologically active isoforms as a consequence of
alternative mRNAs and translational start codon usage. The principal protein products of the PPARG
gene are identified as PPARγ1 and PPARγ2. PPARγ1 is encoded for by exons A1 and A2 then common
exons 1 through 6. PPARγ2 is encoded by exon B and common exons 1 through 6. PPARγ2 is almost
exclusively expressed in adipocytes. Like all nuclear receptors the PPARγ proteins contain a DBD and a
LBD. In addition, like PPARα, the PPARγ proteins contain a ligand-dependent activation function domain
(identified as AF-2) and a ligand-independent activation function domain (identified as AF-1). The AF-2
domain resides in the LBD and the AF-1 domain is in the N-terminal region of the PPARγ proteins.
PPARγ2 protein contains an additional 30 N-terminal amino acids relative to PPARγ1 and these
additional amino acids confer a 5–6-fold increase in the transcription-stimulating activity of AF-1 when
compared to the same domain in the PPARγ1 protein. Expression of PPARγ1 is nearly ubiquitous.
PPARγ2 is expressed near exclusively in white adipose tissue (WAT) where it is involved in lipid storage
and in BAT where it is involved in energy dissipation.
As indicated above, during adipocyte differentiation several upstream genes are required for the
activation of the PPARG gene. These include C/EBPβ and C/EBPδ, SREBP-1c, KLF5, KLF15, zinc-finger
protein 423 (Zfp423), and early B-cell factor (Ebf1). In the process of adipocyte differentiation PPARγ
activates nearly all of the genes required for this process. These genes include aP2 which is required for
transport of free fatty acids (FFAs) and perilipin which is a protein covering the surface of mature lipid
droplets in adipocytes. Additional genes regulated by PPARγ that are involved in lipid metabolism or
glucose homeostasis include lipoprotein lipase (LPL), acyl-CoA synthase (ACS), acetyl-CoA
acetyltransferase 1 (ACAT1), several phospholipase A (PLA) genes, adiponectin, the gluconeogenic
enzyme PEPCK, and glycerol-3-phosphate dehydrogenase (GPD1). PPARγ also functions in macrophage
lipid metabolism by inducing the expression of the macrophage scavenger receptor, CD36. The CD36
receptor is also known as fatty acid translocase (FAT) and it is one of the receptors responsible for the
cellular uptake of fatty acids.
The role of SREBP-1c in activation of adipocyte differentiation is thought to be the result of this
transcription factor initiating the expression of genes that, as part of their activities, generate PPARγ
ligands. This fact explains the necessity for SREBP expression to precede that of PPARγ. In spite of this
fact it has been shown that mice lacking SREBP-1 do not display significant reductions in the amount of
WAT. However, levels of SREBP-2 are increased in these animals indicating that this may be a
compensatory mechanism. Although loss of SREBP-1 expression does not result in a significant deficit in
adipose tissue development, ectopic overexpression of SREBP-1c does enhance the adipogenic activity
of PPARγ.
The C/EBP family of transcription factors were among the first to be shown to play a role in overall
adipocyte differentiation. The three members of the family (C/EBPα, C/EBPβ and C/EBPδ) are highly
conserved basic-leucine zipper containing transcription factors. The importance of these factors in
adipogenesis has been demonstrated in knockout mouse models. for example whole body disruption of
C/EBPα expression results in death shortly after birth due to liver defects, hypoglycemia, and failure of
WAT or BAT accumulation. Using knockout mice it has been determined that the roles of C/EBPβ and
C/EBPδ are exerted early in the process of adiopcyte differentiation whereas those of C/EBPα are
required later. In fact, expression of C/EBPα is induced late in adipogenesis and is most abundant in
mature adipocytes. The expression of both C/EBPα and PPARγ is, in part, regulated by the actions of
C/EBPβ and C/EBPδ. One of the major effects of the expression of C/EBPα in adipocytes is enhanced
insulin sensitivity of adipose tissue. This later fact is demonstrated by the fact that C/EBPα knockout
does not abolish adipogenesis but the WAT is not sensitive to the actions of insulin.
The general model of transcription factor activation of adipogenesis indicates that AP-1, STAT5, KLF4,
and KLF5 are activated early and result in the transactivation of C/EBPβ and C/EBPδ. These latter two
factors in turn activate the expression of SREPB-1 and KLF15 which leads to the activation of PPARγ and
C/EBPα. It is important to keep in perspective that it is not only transcription factor activation of
adipocyte precursors that controls adipogenesis. There is also a balance exerted at the level of
transcription factor-mediated inhibition of adipogenesis. Some of the factors that are anti-adipogeneic
include members of the Krüppel-like factor family, KLF2 and KLF3. GATA2 and GATA3 also exert antiadipogenic activity. GATA factors are so-called because they bind DNA elements that contain a core
GATA sequence. Two of the interferon regulatory factor family of transcription factors, IRF3 and IRF4,
oppose the process of adipogenesis as well.
The changes in the pattern of expression of transcription factors that control the overall process of
adipogenesis is associated with changes in chromatin dynamics. These changes in chromatin dynamics
involve both histone protein methylation and DNA methylation events. The chromatin in pluripotent
cells displays a highly dynamic nature with a high level of decondensed DNA. Once differentiation is
induced there is a change in the overall pattern of methylated genes. Lineage-specific genes are
demethylated whereas pluripotency genes are methylated resulting in transcriptional activation and
silencing, respectively. As the process of adipocyte differentiation proceeds the genes encoding PPARγ
and C/EBPα are observed to be repositioned into the interior of the nucleus coincident with their
increased rates of transcription. Since MSCs can be induced to differentiate into bone and muscle, as
well as adipocyte, it is necessary that adipocyte differentiation genes such as PPARγ and C/EBPα be
silenced if the induced pathway is to bone or muscle.
Associated with transcriptional silencing are protein complexes termed co-repressors and associated
with transcriptional activation are complexes termed co-activators. When MSCs are induced down the
bone lineage the histone 3 proteins in the PPARγ promoter region are methylated on lysine 9 (identified
as H3K9) by a co-repressor complex that includes the histone methyltransferase SETDB1 and the
associated proteins NLK (Nemo-like kinase) and CHD7 (chromodomain helicase DNA binding protein-7).
In addition to silencing of the PPARγ promoter, the activity of the PPARγ protein on its target genes is
also restricted by association with co-repressor complexes. In preadipocytes PPARγ activity is repressed
by association with pRB and HDAC3 (histone deacetylase 3). The induction of differentiation results in
the phosphorylation of pRB which leads to its release from the repressive complex. This in turn results in
the recruitment of histone acetyltransferases (HATs) and the co-activator protein CBP/p300 (CBP is CREB
binding protein, where CREB is cAMP-response element-binding protein) to the PPARγ complex resulting
in activation of PPARγ target gene transcription.
Numerous experiments have begun to define the large array of histone modifications that regulate the
expression of genes involved in overall adipogenesis particularly the expression of PPARγ. These histone
modifying complexes include HATs, HDACs, histone methyltransferases (HMTs), and histone
demethylases (HDMs). The general consequences of activation of HATs and HMTs is the activation of
PPARγ expression and/or enhancement of PPARγ activity at its target gene promoters. Conversely, as
expected, HDAC activation results in inhibited PPARγ activity at its target gene promoters.
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Regulation of Lipid Metabolism in Adipocytes
The triglycerides (TG or TAG) found in WAT represent the major energy reserves of the body. The pool of
TG is in a constant state of flux that is regulated by food intake and fasting and the consequences of
those dietary states on the levels of pancreatic hormones. In addition, adipose tissue fat pools change as
a result of other hormonal fluctuations, inflammatory processes, and pathophysiology. The overall
biochemistry of TG metabolism is covered in the Lipid Synthesis page and the Fatty Acid Oxidation page.
The aim of this section is to discuss in more detail the enzyme activities that regulate overall adipose
tissue TG homeostasis as well as the physiological and hormonal regulation of these processes.
It was originally believed that the liberation of fatty acids from adipose tissue TAG stores was triggered
exclusively via hormonal activation of hormone-sensitive lipase (HSL). However, when HSL-null mice
were generated it was discovered that the process involves additional adipocyte HSL-independent TG
lipase activities. Subsequent research has led to the identification of at least five adipose tissue TAG
lipases in addition to HSL. HSL has demonstrated activity with a wide variety of substrates that includes
TG, diglycerides (DG, also DAG), and cholesterol esters (CEs). When assayed in vitro the activity of HSL is
at least 10-fold higher against DG than TG. When acting on TG or DG the activity of HSL is greatest
against fatty acids that are in the sn-1 or sn-3 position of the glycerol backbone. Until recent
experiments in knock-out mice demonstrated otherwise, HSL was believed to be the principle enzyme
involved in adipocyte TG and DG hydrolysis as well as the primary neutral cholesteryl ester hydrolase
(NCEH) activity.
Although HSL-null mice still exhibit TG hydrolase activity, results from studies in these mice indicate that
HSL-mediated lipolysis is a significant contributor to overall fatty acid liberation from adipocytes. In mice
lacking HSL there is a reduced level of circulating free fatty acids and TGs as well as reduced hepatic
storage of TG. These results indicate that in the absence of HSL there is insufficient adipose tissue
lipolysis to support the normal cellular demands for energy from fatty acids nor for adequate VLDL
synthesis in the liver. Results of studies on the role of HSL in overall adipose tissue lipolysis demonstrate
that it is not strictly required for the initiation of TAG hydrolysis as originally thought. However, in HSLnull mice there is an accumulation of DGs indicating that the critical role for HSL is in the liberation of
fatty acid from DG which in turn generates monoacylglycerides (MG, also MAG). The rate of fatty acid
release from DGs is on the order of 10- to 30-times that the rate of release from TG. To date the only DG
lipase identified in adipose tissue is HSL.
Further understanding of the role of HSL in overall adipose tissue lipolysis came from the identification
of an additional TG lipase that was originally termed desnutrin. The overall structure of desnutrin
indicates that it contains typical domains found in many other lipases. Subsequent to the identification
of desnutrin another lipase was characterized and called adipose triglyceride lipase (ATGL). Desnutrin
and ATGL are the same protein so it is often designated desnutrin/ATGL. The desnutrin/ATGL gene is
expressed predominantly in adipose tissue but also at much lower levels in cardiac and skeletal muscle
and the testes. The intracellular location of desnutrin/ATGL is the cytosol as well as in tight association
with lipid droplets. The activity of desnutrin/ATGL is specific for TG as evidenced in cell culture
experiments where over-expression of the gene results in increased free fatty acid release with no effect
on phospholipid stores. In addition, desnutrin/ATGL has limited activity against DG since in these and
similar in vitro experiments there is a significant accumulation of DG compared to the same types of
experiments carried out with HSL.
Expression of desnutrin/ATGL is under the influence of dietary status. In fasting animals the level of
desnutrin/ATGL increases and then declines following re-feeding. This dietary regulation of
desnutrin/ATGL suggests that it may play a contributory role in the development of obesity, a
hypothesis supported by the fact that in genetically obese mice (ob/ob and db/db) the level of
desnutrin/ATGL expression is reduced. When experiments are performed that artificially reduce the
level of desnutrin/ATGL RNA or protein there is a significant drop in the level of free fatt acid release.
Demonstrating a synergy between HSL and desnutrin/ATGL activity, in cells where both enzymes are
reduced there is an additive level of reduction in free fatty acid release. A critical role for
desnutrin/ATGL in TG hydrolysis in tissues other than adipose tissue was shown by results of
desnutrin/ATGL knock-out in mice. These animals died at around 12-weeks of age due to increased
ectopic fat stores particularly in the heart. In addition, total lipase activities in several tissues in addition
to WAT and BAT were altered in the desnutrin/ATGL-null mice. These data point to a critical role for
desnutrin/ATGL in TG hydrolysis and fatty acid release not only from adipose tissue but also from tissues
such as the heart, skeletal muscle, and testes.
In addition to HSL and desnutrin/ATGL, adipose tissue expresses a number of other TG hydrolases.
Adipose tissue microsomes contain a non-HSL TG lipase that is identified as triacylglycerol hydrolase
(TGH, also called carboxylesterase 3). TGH contains typical lipase motifs and displays catalytic activity
against long-, medium-, and short-chain TAGs as well as neutral cholesteryl esters. However, TGH does
not hydrolyze phospholipids . Expression of TGH is seen predominantly in the liver where its primary
functions are to mobilize intracellular TAG stores and participate in the synthesis of TG-rich VLDLs. TGH
expression is also seen in adipocytes and the level of its expression increases dramatically when
preadipocytes differentiate into mature adipocytes. The adipose tissue regulation of TGH expression is
effected, in part, via the action of C/EBPα. A related protein, identified as TGH-2, has also been found
predominantly expressed in the liver but is also present in adipose tissue and kidney.
There is another interesting protein expressed predominantly in adipose tissue with a significant degree
of homology to desnutrin/ATGL. This protein is called adiponutrin. Whereas, adiponutrin shows TG
lipase activity when assayed in vitro, when it is over-expressed in cells it has no effect on TG hydrolysis.
In addition, whereas desnutrin/ATGL (as well as most other lipases) expression is increased in the fasting
state and decreased following re-feeding, adiponutrin expression exhibits the opposite pattern. In fasted
animals adiponutrin mRNA is essentially undetectable and its levels increase dramatically in the re-fed
state. It appears that although this enzyme is a member of the lipase family of enzymes it plays an
anabolic rather than a catabolic role in adipocyte lipid metabolism.
The final step in the complete hydrolysis of TGs occurs when glycerol and the last fatty acid are released
from MGs by MAG lipase. This enzyme possesses no catalytic activity towards TGs or DGs nor cholesteryl
esters. Numerous other proteins in addition to the lipases are involved in overall TG homeostasis in
adipose tissue. Several of these other proteins are associated with the lipid droplets inside the cell such
as the perilipins, adipose fatty acid-binding protein (aP2, also called FABP4 and aFABP), and caveolin-1.
Additional proteins important in overall TG metabolism include aquaporin 7 (a water and glycerol
transport protein) and lipotransin. The perilipins play a role in restricting access of TG lipases to
substrates in order to prevent unrestrained hydrolysis in the un-stimulated state. The role of aP2 is to
carry free fatty acids from the fat droplet to the plasma membrane where they can be released to the
plasma. The glycerol that is released from TGs is exported via the action of aquaporin 7 as shown by
experiments in mice lacking expression of this gene. These mice release free fatty acids upon stimulation
of adipose tissue with catecholamines but no glycerol is released. The role of lipotransin is believed to
be in shuttling HSL from the cytosol to the lipid droplet upon stimulation of adipocytes.
Whether adipose tissue stores fatty acids as TGs or releases them for energy production by other tissues
is dependent upon the dietary, hormonal and physiological status of the organism. The primary
mechanism for the stimulation of adipose tissue TG hydrolysis is discussed in the fatty acid oxidation
page. In brief, catecholamines such as epinephrine and norepinephrine, as well as the pancreatic
hormone glucagon, bind to their cognate receptors on adipocytes triggering activation of adenylate
cyclase resulting in increased levels of cAMP. In turn the cAMP activates PKA which then phosphorylates
and activates HSL. Of significance is the fact that activation of PKA in HSL-null mice also results in
enhanced TG hydrolysis albeit at a level much lower than in the presence of active HSL. This indicates
that there are PKA-mediated events in adipose tissue TG lipolysis that are distinct from the classic HSLmediated process.
The primary change in adipose tissue TG lipolysis following feeding is exerted via the actions of insulin.
The cAMP-dependent changes that occur in response to insulin binding are effected by activation of
phosphodiesterase 3B which hydrolyzes cAMP rendering PKA much less active. The activation of
phosphodiesterase 3B occurs via PKB/Akt-mediated phosphorylation which itself is activated following
insulin binding its receptor. The principle cAMP-independent mechanism for insulin-mediated reduction
in TG lipolysis is due to the stimulation of protein phosphatase-1 which removes the phosphate from
HSL rendering it much less active. The activity of HSL is also affected via phosphorylation by AMPK. In
this case the phosphorylation inhibits the enzyme. Inhibition of HSL by AMPK may seem paradoxical
since the release of fatty acids stored in TGs would seem necessary to promote the production of ATP
via fatty acid oxidation and the major function of AMPK is to shift cells to ATP production from ATP
consumption. This paradigm can be explained if one considers that if the fatty acids that are released
from TGs are not consumed they will be recycled back into TGs at the expense of ATP consumption.
Thus, it has been proposed that inhibition of HSL by AMPK mediated-phosphorylation is a mechanism to
ensure that the rate of fatty acid release does not exceed the rate at which they are utilized either by
export or oxidation.
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Table of Adipose Tissue Hormones and Cytokines
Adipose tissue produces and releases a vast array of protein signals including growth factors, cytokines,
chemokines, acute phase proteins, complement-like factors, and adhesion molecules. The Table below
describes several of the adipocyte proteins in more detail with leptin, adiponectin, and resistin discussed
in greater detail in the following sections. The proteins of the various signaling processes are listed
below. Not all WAT secretes the same adipokines as is evident from studies of differences in adipose
tissue function in various anatomical regions of the body as described above. This is most significant
when considering the clinical risks associated with increased adipose tissue mass. For example, increases
in visceral WAT, even when subcutaneous fat depots are not increased, carry a greater metabolic risk for
insulin resistance and diabetes and cardiovascular disease.
Growth and angiogenic factors: fibroblast growth factors (FGFs), insulin-like growth factor-1 (IGF-1),
hepatocyte growth factor (HGF), nerve growth factor (NGF), vascular endothelial cell growth factor
(VEGF), transforming growth factor-β (TGFβ), angiopoietin-1, angiopoietin-2, tissue factor (TF, factor 3)
Cytokines: IL-1β, IL-4, IL-6, IL-8, IL-10, IL-18, TNFα, macrophage migration inhibitory factor (MIF)
Complement-like factors: adipsin, adiponectin, acylation stimulating protein (ASP)
Adhesion molecules and ECM components: α2-macroglobulin, vascular cell adhesion molecule-1
(VCAM-1), intercellular adhesion molecule-1 (ICAM-1), collagen I, collagen III, collagen IV, collagen VI,
fibronectin, matrix metalloproteinase 1 (MMP1), MMP7, MMP9, MMP10, MMP11, MMP14, MMP15,
lysyl oxidase
Acute phase proteins: C-reactive protein (CRP), serum amyloid A3 (SAA3), plasminogen activator
inhibitor-1 (PAI-1), haptoglobin
Chemokines: chemerin, monocyte chemotactic protein-1 (MCP-1), macrophage inhibitory protein-1
alpha (MIP-1α), regulated upon activation, normally T cell expressed and secreted (RANTES)
Metabolic processes: adipocyte fatty acid binding protein (aP2), apolipoprotein E (apoE), resistin,
omentin, vaspin, apelin, retinol binding protein 4 (RBP4), visfatin, leptin
Other processes: COX pathway products PGE2 and PGI2 (prostacyclin), nitric oxide synthase pathway,
renin-angiotensin system, tissue inhibitors of metalloproteinases (TIMPs)
In addition to these secreted factors adipose tissue produces several plasma membrane and nuclear
receptors that can trigger changes in adipose tissue function. Plasma membrane receptors include those
for insulin, glucagon, growth hormone, adiponectin, gastrin, and angiotensin-II. Nuclear receptors
include peroxisome proliferator-activated receptor-γ (PPARγ), estrogens, androgens, vitamin D, thyroid
hormone, progesterone, and glucocorticoids.
Factor
Principal Source
Major Action
adiponectin
also called adipocyte complement
factor 1q-related protein (ACRP30),
and adipoQ
adipocytes
see below
adipocytes, liver,
adipsin (also called complement factor
monocytes,
D)
macrophages
rate limiting enzyme in complement
activation
apelin
levels increase with increased insulin,
exerts positive hemodynamic effects, may
adipocytes, vascular
regulate insulin resistance by facilitating
stromal cells, heart
expression of BAT uncoupling proteins (e.g.
UCP1, thermogenin)
adipocytes, liver
modulates expression of adipocyte genes
involved in glucose and lipid homeostasis
such as GLUT4 and fatty acid synthase
(FAS); potent anti-inflammatory effects on
macrophages expressing the chemerin
receptor (chemokine-like receptor-1,
CMKLR1)
C-reactive protein (CRP)
hepatocytes,
adipocytes
is a member of the pentraxin family of
calcium-dependent ligand binding proteins;
assists complement interaction with
foreign and damaged cells; enhances
phagocytosis by macrophages; levels of
expression regulated by circulating IL-6;
modulates endothelial cell functions by
inducing expression of various cell
adhesion molecules, e.g. ICAM-1, VCAM-1,
and selectins; induces MCP-1 expression in
endothelium; attenuates NO production by
downregulating NOS expression; increase
expression and activity of PAI-1
IL-6
adipocytes,
hepatocytes,
acute phase response, B cell proliferation,
activated Th2 cells,
thrombopoiesis, synergistic with IL-1 and
and antigenTNF on T cells
presenting cells
(APCs)
leptin
predominantly
adipocytes,
mammary gland,
intestine, muscle,
placenta
chemerin
see below
leukocytes,
adipocytes
is a chemokine defined as CCL2 (C-C motif,
ligand 2); recruits monocytes, T cells, and
dendritic cells to sites of infection and
tissue injury
omentin
visceral stromal
vascular cells of
omental adipose
tissue
the omentum is one of the peritoneal folds
that connects the stomach to other
abdominal tissues, enhances insulinstimulated glucose transport, levels in the
blood inversely correlated with obesity and
insulin resistance
plasminogen-activator inhibitor-1
(PAI-1)
adipocytes,
monocytes,
see the Blood Coagulation page for more
placenta, platelets, details
endometrium
resistin
adipocytes, spleen,
monocytes,
macrophages, lung, see below
kidney, bone
marrow, placenta
monocyte chemotactic protein-1
(MCP-1)
TNFα
primarily activated
macrophages,
adipocytes
induces expression of other autocrine
growth factors, increases cellular
responsiveness to growth factors and
induces signaling pathways that lead to
proliferation
vaspin
visceral and
subcutaneous
adipose tissue
is a serine protease inhibitor, levels
decrease with worsening diabetes,
increase with obesity and impaired insulin
sensitivity
visfatin; also called pre-B cellenhancing factor (PBEF);
visceral white
reported to be the extracellular
adipose tissue
version of the enzyme nicotinamide
phosphoribosyltransferase (Nampt or
eNampt), however, the original paper
conflicting results relative to insulin
receptor binding but blocking insulin
receptor signaling interferes with effects of
eNampt; changes in eNampt activity occur
during fasting and positively regulate the
activity of the NAD+-dependent
deacetylase SIRT1 leading to alterations in
claiming this has been retracted
gene expression
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Leptin
Leptin is 16kDa peptide whose central function is the regulation of overall body weight by limiting food
intake and increasing energy expenditure. However, leptin is also involved in the regulation of the
neuroendocrine axis, inflammatory responses, blood pressure, and bone mass. The human leptin gene is
the homolog of the mouse "obese" gene (symbol OB) that was originally identified in mice harboring a
mutation resulting in a severely obese phenotype. Leptin-deficient (ob/ob) and leptin receptor-deficient
(db/db) mice exhibit numerous disruption in energy, hormonal, and immune system balance. These
mice are obese, display hormonal imbalances, have defects in thermoregulation, have hematopoietic
defects and are infertile. Levels of leptin increase in the serum in obese individuals and drop during
weight loss. There is a direct correlation between the amount of body fat an individual carries and the
circulating levels of leptin. Leptin activates the anorexigenic axis (appetite suppression) in the arcuate
nucleus (ARC) of the hypothalamus by increasing the frequency of action potentials in the hypothalamic
POMC neurons by depolarization through a nonspecific cation channel and by reduced inhibition by
local orexigenic neuropeptide-Y (NPY) neurons.
Leptin functions by binding to its receptor which is a member of the cytokine receptor family. Leptin and
its receptors possess structural similarities to the IL-6 family of cytokines and the class I cytokine
receptor family. The leptin receptor mRNA is alternatively spliced resulting in six different products. The
leptin receptors are named Ob-R, OB-Rb, OB-Rc, Ob-Rd, Ob-Re, and Ob-Rf. The OB-Rb mRNA encodes
the long form of the leptin receptor (also called LEPR-B) and is expressed primarily in the hypothalamus
but is also expressed in cells of the innate and adaptive immune systems as well as in macrophages. The
other receptor subtypes are expressed in numerous tissues including muscle, liver, kidney, adrenal
glands, leukocytes, and vascular endothelium. Activation of the receptor leads to increased
phosphatidylinositol-3-kinase (PI3K) and AMPK activity via activation of the Jak/STAT signaling pathway.
One effect of the activation of the Jak/STAT pathway is activation of suppressor of cytokine signaling 3
(SOCS3) which then inhibits leptin signaling in a negative feed-back loop. Leptin binding its receptor also
results in the activation of mTOR both in the hypothalamus and in peripheral tissues. The role of mTOR
in the regulation of protein synthesis is covered in both the Protein Synthesis page as well as in the
Insulin Functions page. The role of leptin in the activation of mTOR function is an important factor in the
ability of leptin to activate macrophages. An interesting aspect of the role of leptin in mTOR function is
that within mature adipocytes leptin synthesis itself is dependent on mTOR activation. Given that leptin
levels rise in the serum of obese individuals and that leptin interaction with macrophages leads to
increased macrophage inflammatory processes, it is not surprising that there is a direct correlation
between leptin levels and the development of atherosclerosis.
When leptin binds to its receptor (LEPR-B) the receptor undergoes a conformational change that
activates the receptor-associated Jak2 tyrosine kinase. Activated Jak2 will autophosphrylate itself as well
as phosphorylate the tyrosine (Y) residues in LEPR-B at positions 985, 1077, and 1138. Phosphorylated Y985 serves as a docking site for SHP2 (SH2 domain containing protein tyrosine phosphatase, also called
PTP1D). The gene that encodes SHP2 is identified as PTPN11. Phosphorylated Y-1077 serves as a docking
site for STAT5 (signal transduction and activation of transcription 5). Phosphorylated Y-1138 serves as a
docking site for STAT3. When SHP2, STAT5, and STAT3 bind to phosphorylated LEPR-B they themselves
are activated by Jak2-mediated phosphorylation. Activated SHP2 in turn activates the ERK1/2
(extracellular-regulated kinase 1/2) signal pathway that results in increased transcription of the EGR-1
gene. Activated STAT3 in turn activates the transcription of SOCS3 (suppressor of cytokine signaling 3).
SOCS3 will then interact with Y-985 and attenuate signaling from SHP2 as well as interact with Jak2 and
attenuate its tyrosine kinase activity resulting in a negative feed-back loop.
Leptin expression is under complex control and a number of transcription factor binding sites have been
identified in the promoter region of the leptin gene. Leptin levels are higher in age and weight-matched
females compared with males. This is partially due to the inhibition of leptin expression by androgens
and the stimulation of expression by estrogens. Leptin expression has been shown to be increased by
sex steroids, glucocorticoids, cytokines, and toxins released during acute infection. The sympathetic
nervous system triggers a reduction in circulating leptin levels via the release of catecholamines. This
effect of catecholamines has been shown to be due to activation of β-adrenergic receptor signaling.
In addition to effects on appetite exerted via central nervous system functions, leptin is also known to
exert effects on inflammatory processes. Leptin modulates peripheral T cell function leading to
increased levels of T helper cell type 1 cytokines. In addition leptin reduces thymocyte apoptosis and
increases thymic cellularity. These result correlate well with observations demonstrating a reduced
capacity for immunologic defense when leptin levels are low. However, too much leptin is not beneficial
as high concentrations can result in an abnormally strong immune response which predisposes an
individual to autoimmune phenomena. Acute stimulation with pro-inflammatory cytokines results in
increased serum levels of leptin, whereas, chronic stimulation by IL-1, IL-6, or TNFα leads to reduced
levels of serum leptin.
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Adiponectin
Adiponectin was independently isolated by four different laboratories leading to different names.
However, adiponectin is considered the standard name for this adipose tissue-specific protein. Other
names include adipocyte complement related protein of 30kDa (ACRP30) because of its homology to
complement factor 1q (C1q), adipoQ, gelatin-binding protein 28kDa (BGP28), and adipocyte most
abundant gene transcript 1 (apM1). The major biological actions of adiponectin are increases in insulin
sensitivity and fatty acid oxidation.
The adiponectin gene is located on chromosome 3q27.3 spanning 16 kbpand composed of 3 exons. The
adiponectin gene does not contain a typical typical TATA-box promoter element upstream of the
transcriptional start site. Adiponectin contains a C-terminal globular domain which harbors the
homology to C1q and an N-terminal collagen-like domain. The globular domain allows for a
homotrimeric association of the protein forming the functional structure of the protein. The association
of the subunits is such that two trimeric globular domains interact with a single stalk of collagen
domains formed from two trimers. This complex structure is similar to the TNF superfamily of proteins
despite there being no amino acid sequence homology between adiponectin and TNF proteins. Within
the circulation, adiponectin exists in both a full-length form as well as a globular form that is the result
of proteolytic cleavage of the full-length protein. The hormone forms complex structures such that it can
be found as a trimer, hexamer, and as the high molecular weight oligomer. In addition to the complex
structure, adiponectin is glycosylated, a modification that is essential to its activity.
Adiponectin activity is inhibited by adrenergic stimulation and glucocorticoids. Expression and release of
adiponectin is stimulated by insulin and inhibited by TNF-α. Conversely, adiponectin exerts inflammatory
modulation by reducing the production and activity of TNF-α and IL-6. Unlike leptin, levels of
adiponectin are reduced in obese individuals and increased in patients with anorexia nervosa. There are
sex-related differences in adiponectin levels as well similar to that seen for leptin where age and weight
matched males have lower levels than females. In patients with type 2 diabetes, levels of adiponectin
are significantly reduced.
Adiponectin functions by interaction with specific cell-surface receptors and at least two receptors have
been identified. AdipoR1 is expressed at highest levels in skeletal muscle and AdipoR2 in primarily
expressed in the liver. Expression of adiponectin receptors is also seen in various regions of the brain.
AdipoR1 is highly expressed in the medial prefrontal cortex, hippocampus, and the amygdala. Whereas,
AdipoR2 expression in the brain is restricted to the hippocampus and specific hypothalamic nuclei.
AdipoR1 is a high-affinity receptor for globular adiponectin and has low affinity for the full-length
adiponectin. In contrast, AdipoR2 has intermediate affinity for globular and full-length adiponectin.
Although these receptors contain seven transmembrane domains typical of the serpentine family of Gprotein coupled receptors (GPCRs), they are structurally distinct from the GPCR class. The AdipoRs
stimulate the phosphorylation and activation of AMPK. The adiponectin-mediated activation of AMPK
results in increased glucose uptake, increased fatty acid oxidation, increased phosphorylation and
inhibition of acetylCoA carboxylase (ACC) in muscle. In the liver the result is reduced activity of
gluconeogenic enzymes and glucose output.
Adiponectin also plays an important role in hemostasis by suppressing TNFα-mediated inflammatory
changes in endothelial cell responses and inhibiting vascular smooth muscle cell proliferation. Activation
of AMPK activity in endothelial cells results in increased fatty acid oxidation and activation of endothelial
NO synthase (eNOS).
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Resistin
Resistin is a 12 kDa protein that was originally identified in mice in a screen for genes suppressed by an
agonist of the peroxisome proliferator-activated receptor-γ (PPARγ). The name resistin derives from the
original observation that this protein induced insulin resistance in mice. Resistin belongs to a family of
four proteins referred to as FIZZ proteins for "found in inflammatory zone". Resistin is thus, also referred
to as FIZZ3. Although resistin is expressed in adipocytes, in humans it appears that macrophages may be
the most important source of the protein.
In mice resistin expression increases during adipocyte differentiation and levels of resistin increase in
diet-induced obesity. Reduction in resistin levels is associated with increased AMPK activity in the liver
which leads to decreased expression of gluconeogenic enzymes and consequent reduction in hepatic
glucose production. Conversely, elevation in resistin levels is associated with increased hepatic glucose
production and glucose intolerance. Whether these same responses to resitin are evident in human is
still under investigation. What is known is that overexpression of resistin in human heptocytes impairs
insulin-stimulated glucose uptake and glycogen synthesis. Part of the mechanism for impaired glycogen
synthesis is that resistin decreases the expression of one of the insulin receptor substrates (IRS-2) which
is involved in the activation of PI3K. The PI3K-activated signal pathway leads to the phosphorylation and
inhibition of glycogen synthase kinase 3β (GSK3β). Un-phosphorylated GSK3β would normally
phosphorylate and inhibit glycogen synthase activity. Loss of this pathway then leads to a higher rate of
glycogen synthase inhibition by GSK3β-mediated phosphorylation.
Resistin also exerts effects on the immune system and the vasculature. Resistin modulates endothelial
cell function by enhancing expression of the cell adhesion molecule VCAM-1 and the chemoattractant
MCP-1. Resistin has also been shown to exert a pro-inflammatory effect on smooth muscle cells.
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Inflammatory Functions of Adipose Tissue
The significance of inflammatory responses elicited via secretion of adipose tissue-derived (WAT)
cytokines relates to the fact that their production and secretion is increased in obese individuals. There
is a growing body of evidence that demonstrates a direct link between the changes in adipose tissue
function in obesity and the development of type 2 diabetes and the metabolic syndrome. One key
change in adipose tissue during obesity is an increase in the percentage of macrophages resident within
the tissue. Macrophages are a primary source of pro-inflammatory cytokines secreted by adipose tissue.
The primary adipokine responsible for this infiltration is monocyte chemotactic protein-1 (MCP-1).
As the level of macrophages increases in adipose tissue the level of pro-inflammatory cytokine secretion
by the tissue increases. Circulating levels of both TNF-α and IL-6 increase as adipose tissue expands in
obesity and these changes are directly correlated with insulin resistance and the development of type 2
diabetes. As indicated above adiponectin plays an important anti-inflammatory role by suppressing the
production of both TNF-α and IL-6. However, as the level of macrophage infiltration increases in obesity
the increased secretion of TNF-α results in suppression of adiponectin production and secretion.
Adipose tissue-derived IL-6 accounts for approximately 30% of the circulating level of this proinflammatory cytokine. Visceral WAT has been shown to secrete a higher percentage of the circulating
IL-6 than subcutaneous WAT and this fact correlates with the negative effects of a pro-inflammatory
status (as is the case with obesity) on the organs. As WAT density increases there is an associated
increase in IL-6 secretion which is correlated to an increase in the circulating levels of acute-phase
proteins such as CRP. In addition to the effects of TNF-α on adiponectin production the cytokine also
directly affects insulin sensitivity by inhibiting insulin receptor signaling. This effect of TNF-α is the result
of increased insulin receptor serine phosphorylation. TNF-α (as well as IL-6) trigger the release of proinflammatory cytokines such as JUN N-terminal kinase (JNK) and nuclear factor kappa B (NFκB).
Activation of JNK leads to the insulin receptor serine phosphorylation as well as insulin receptor
substrate (IRS) serine phosphorylation. Both of these TNF-α-mediated serine phosphorylations lead to
impaired insulin receptor signaling. JNK and NFκB activate pro-inflammatory genes which results in a
self-perpetuating cycle of increased inflammatory cytokine release. TNF-α also decreases endothelial
nitric oxide synthase (eNOS) resulting in decreased levels of NO as well as decreased expression of
mitochondrial oxidative phosphorylation genes. This leads increased oxidative stress, accumulation of
reactive oxygen species (ROS), and increased endoplasmic reticulum stress. The responses of the cell to
the increased oxidative stress is further increases in NFkB releases and thus, increased inflammatory
processes. The effects of adipocyte-derived TNF-α and IL-6 demonstrates a clear correlation between
obesity and a pro-inflammatory role for adipocytes.
Although lymphocytes (T-cells and B-cells) are not constituents of adipose tissue they are physically
associated within the lymph nodes. Lymph tissue is surrounded by pericapsular adipose tissue which
increases in density with increasing obesity. This close association allows for 2-way paracrine
interactions between the lymph and adipose tissues. One important interaction between lymph tissue
and WAT involves leptin. As indicated above, leptin plays a major role in the regulation of appetite and
energy balance and the circulating levels of leptin increases as adipose tissue mass increases. Leptin has
also been shown to modulate T-cell function, thus demonstrating a pro-inflammatory role for leptin.
Leptin protects T-cells from apoptosis and enhances the switching of T-cells to a Th1 response. Proinflammatory cytokine production and release from T-cells is increased as a result of leptin action. Leptin
induces a number of signal transduction pathways in immune and endothelial cells as outlined above.
Leptin effects on the vascular endothelium are also pro-inflammatory. Expression of adhesion molecules
is increased by leptin binding its receptor on endothelial cells. This results in an increased ability of
neutrophils and other leukocytes to adhere to the endothelium leading to increased local inflammatory
processes.
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Metabolic Functions of Brown Adipose Tissue, BAT
It was originally thought that BAT was present in humans only during the neonatal period. However,
recent evidence has demonstrated that adults retain some metabolically active BAT deposits that
respond to cold and sympathetic nervous system activation. The most studied regulator of the action of
BAT is norepinephrine.
Within BAT, norepinephrine interacts with all three types of adrenergic receptors (α1, α2, and β) each of
which activates distinct signaling pathways in the brown adipocyte. With respect to the β-
adrenoreceptors, the β3 subtype is the most significant, β1 receptors are expressed in brown
preadipocytes but not mature adipocytes and β2 receptors are expressed in BAT but not brown
adipocytes themselves. Of significance to the role of BAT in thermogenesis and the major role of β3adrenoreceptors is the fact that these receptors are not subject to desensitization as are the β1 and β2
receptors. However, this is not to say that continuous adrenergic stimulation has no negative effect in
BAT. Indeed, the level of β3 receptor expression is downregulated during continuous adrenergic
stimulation. However, this effect is transient and the mRNA rapidly re-accumulates after termination of
the stimulatory signal.
Signal transduction events triggered by adrenergic stimulation of BAT are effected via the activation of
Gs type G-proteins. Gs proteins couple to the activation of adenylate cyclase resulting in increased cAMP
production and activation of PKA. For more information on the types of G-proteins visit the Signal
Transduction page. Activation of HSL leads to release of free fatty acids which are taken up by the
mitochondria similarly as they would be for purposes of oxidation, however, in BAT they interact with
and activate the proton gradient uncoupling activity of uncoupling protein 1 (UCP1, also known as
thermogenin). Uncoupling the proton gradient releases the energy of that gradient as heat. The
thermogenic function of BAT is outlined in the Figure below. In addition to stimulating heat production
in BAT, norepinephrine promotes the proliferation of brown preadipocytes, promotes the differentiation
of mature brown adipocytes, inhibits apoptosis of brown adipocytes, and regulates the expression of the
UCP1 gene.
Hormonal generation of heat in brown fat: In response to cold norepinephrine binds to β3-adrenergic
receptors on browen adipocytes resulting in the activation of adenylate cyclase. Active adenylate cyclase
generates cAMP which in turn activates PKA leading to phosphorylation and activation of HSL, Active
HSL then accelerates fatty acid release from stored triglycerides. Some of the fatty acids are oxidized
and a few molecules of fat activate UCP1 which uncouples the proton gradient in the mitochondria
releasing the energy as heat.
Interestingly, norepinephrine stimulation of α2-adrenergic receptors on BAT has the opposite effect to β3
receptor activation. This is due to the fact that the α2 receptors are coupled to Gi type G-proteins whose
activation results in inhibition of adenylate cyclase. The precise physiological significance of this parallel
response of BAT to norepinephrine is as yet not fully appreciated. It is unclear how, or when, BAT
balances the thermogenic stimulatory actions of β3 receptors with the inhibitory actions of α2 receptors
but it may allow the cells to modulate their responses to norepinephrine under varying physiological
conditions.
Similar to the endocrine role of WAT, BAT also synthesizes and secretes numerous factors. However,
due to the relatively small overall size of brown fat compared to white fat the role of factors secreted
from BAT serve many autocrine and paracrine roles. This is not to say that BAT doesn't have a potential
endocrine role it is just not as well established as the endocrine role of WAT.
Several proteins produced from BAT serve autocrine functions such as adipsin, fibroblast growth factor 2
(FGF2), insulin-like growth factor-1 (IGF-1), prostaglandins, and adenosine. Adipsin (also known as
complement factor D) cleaves the complement protein C3 into C3a and C3b. C3a is inactivated to
C3desArg which is more commonly referred to as acylation-stimulating protein (ASP). ASP stimulates
TAG synthesis and glucose uptake in WAT. The level of C3 expression is much higher in WAT than in BAT
and ASP has not been found in BAT so the precise function of adipsin in BAT is not clear although it could
be functional when BAT is in a thermogenically inactive state and is functioning in an anabolic state to
store TAGs. Acute and chronic cold exposure results in increased FGF2 expression in BAT as does
norepinephrine. The FGF receptor 1 (FGFR1) gene is also expressed in BAT and the result of FGF2
activation of the receptor is increased brown adipocyte cell density. Similarly, during cold exposure the
levels of IGF-1 mRNA increase and IGF-1 receptors are highly expressed on brown adipocytes. IGF-1
action can prevent TNF-α-induced apoptosis in brown fat.
Paracrine factors synthesized by BAT include nerve growth factor (NGF), vascular endothelial cell growth
factor (VEGF), angiotensinogen, and NO. The secretion of NGF occurs primarily from proliferating brown
preadipocytes and in this capacity is believed to promote sympathetic innervation of the tissue which in
turn permits increased norepinephrine stimulation of the cells in BAT. Expression of VEGF is high in
proliferating and mature brown adipocytes and the VEGF receptors, FLK-1 and FLK-4, are expressed in
BAT. The expression of VEGF in BAT may promote and maintain the high level of vascularization in this
tissue. Norepinephrine stimulation and cold stress both result in increased levels of VEGF expression in
BAT. Both inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) are expressed in BAT. In
addition to its expression in the endothelial cells of BAT, eNOS expression is seen in brown adipocytes.
Norepinephrine stimulation of BAT, as well as brown adipocytes in culture, results in increase NO
production. Within brown adipocytes the production of NO may lead to inhibition of mitochondrial
oxidation. Within BAT the production of NO likely promotes rapid increases in blood flow.