Download LDL

PPARs
number of publications
PPAR – history of research
3000
2000
1000
0
1985
1990
1995
year
2000
2005
fibroblasts with peroxisomes stained in green
Peroxisomes
• The ubiquitous organelles, which are delineated by a single membrane, generally
contain enzymes that utilize oxygen to subtrate hydrogen atoms from certain organic
substrates in an oxidative reaction that generates hydrogen peroxide.
• Peroxisomes also typically contain catalase, an enzyme that uses this toxic byproduct
of metabolism to oxidize formic acid, alcohols, phenols, and other substrates. Any
remaining hydrogen peroxide present in the cell is broken down by catalase into water
and free oxygen molecules.
• The degradation of fatty acids and the catalysis of the initial steps in the synthesis of
ether phospholipids, which are eventually utilized in membrane formation, are a few of
the other various tasks commonly carried out by peroxisomes.
peroxisome proliferators
• Peroxisome proliferators are a diverse
group of chemicals.
• Characteristic responses of rodent
hepatocytes to peroxisome proliferators
include hepatomegaly, proliferation of
peroxisomes in parenchymal cells, and an
increase in peroxisomal β-oxidation of fatty
acids.
• This elevated capacity to catabolize fatty
acids is attributed to induction of all
enzymes in the peroxisomal β-oxidation
cascade, in particular the first and ratelimiting enzyme, acyl-CoA oxidase (ACO).
• The peroxisome proliferator-response has
gained considerable interest due to its
association with metastatic hepatocellular
carcinomas in rodents. The mechanism by
which peroxisome proliferators cause
cancer is not clear - these compounds do
not bind directly and damage DNA.
PPARs (peroxisome proliferator-activated receptors)
• PPARs were cloned in 1990 as transcription factors that mediate the effects of
synthetic peroxisome proliferators, the molecules known to stimulate proliferation of
peroxisomes in rodents.
• Since this time PPARs have been described in a wide variety
of species ranging from zebrafish and Xenopus to mouse and
human.
• The ligand binding pocket of PPARs is much larger than
that of other NRs, with a volume of 1300 Å, of which the
ligand occupies only about 30% to 40%.
Xenopus laevis
• Overall, PPARs appear to have evolved as NRs adapted for binding to multiple
natural ligands with relatively low affinity.
• The first PPRE was identified in the promoter of the acyl coenzyme A (acyl-CoA)
oxidase gene, and then in a number of genes known to be transcriptionally activated
during adipocyte differentiation or associated with lipid metabolism.
PPARs (peroxisome proliferator-activated receptors)
- Lipid-activated transcription factors
- Regulate:
* lipid metabolism
* glucose homeostasis
- Impaired PPAR activity is believed to lead to dyslipidemia and insulin resistance
Regulation of PPAR activity
- Availability of ligands
- Availability of 9cis-retinoic acid (PPAR-RXR
is a permissive dimer)
- Availability of cofactors
- PPARs bind NCoR and SMRT but do not repress target genes in the absence
of their ligands
- PPARs interact with coactivators of p160 class, p300, CBP and DRIP.....
(different ligands selectively induce the requirement of different cofactors)
- Phosphorylation
- insulin induces phosphorylation of PPARα and PPARγ leading to increased
transcriptional activity
- some growth factors through MAP kinases phosphorylate PPARγ and PPARα
leading to decreased transcriptional activity
- Ubiquitination
- ligand binding can induce ubiquitination and proteasome degradation of PPARγ
PPAR subtypes
• In mammals, PPAR family consists of three subtypes of proteins encoded by separate
genes (PPARα, PPARβ, PPARγ) with varying degrees of homology. For instance, human
PPARγ and Xenopus PPARγ are more closely related in terms of amino acid identity than
human PPARα and human PPARγ.
• All PPAR subtypes share a high degree of amino acid sequence similarity, both within
their DBD and LBD domains. This is reflected in functional similarities in that these
receptors are activated by structurally related compounds.
• Nevertheless, the three PPAR subtypes appear to serve distinct roles in vivo. They
exhibit markedly different tissue distributions, have different affinities for different
PPREs, and are activated to different extents and by different ligands.
• A most fascinating finding is that the two isotypes α and γ have balanced regulatory
actions in fatty acid oxidation in the liver via PPARα, and fatty acid storage in the adipose
tissue via PPARγ.
• PPARs can also repress gene transcription by antagonizing the NFκB, AP-1, NFAT....,
signaling pathways, independently of DNA binding, via protein-protein interactions that
lead to formation of inactive complexes and attenuating inflammation.
PPAR comprises three proteins:
- PPARα
* predominantly expressed in the liver, kidney, heart, skeletal muscle
* controls FA catabolism
* expressed in macrophages and foam cells
- PPARβ (PPARδ)
* ubiquitously expressed
* controls lipid metabolism in the brain and heart
* regulates differentiation of some type of cells, e.g. keratinocytes
- PPARγ
* predominantly expressed in the brown and white adipose tissue, in intestine,
macrophages, retina, etc.
* controls lipid metabolism and promotes lipid storage
* induces differentiation and maturition of adipocytes
* modulates action of insulin
* expressed in macrophages and foam cells
PPARα
• PPARα was the first of PPARs cloned and characterized.
• The levels of PPARα are highest in the brown adipose tissue and in the liver, then come
the heart, kidney, and enterocytes, but its expression has also been detected in many
other cell types including monocytes, endothelium and vascular smooth muscle cells.
• Endogenous PPARα ligands are some fatty acids and their derivatives, like 8(S)hydroxyeicosatetranoic acid (8(S)-HETE) and leukotriene B4 (LTB4).
• Importantly, PPARα isotype is the
bezafibrate, and fenofibrate, which
treatment of cardiovascular diseases.
cellular target for
are hypolipidemic
fibrates such as gemfibrozil,
drugs widely used for the
PPARα-/- mice
• Overall, PPARα acts as a global regulator of energy metabolism, which coordinates the
rates of utilization of the various energy sources in relation to food availability.
• Accordingly, PPARα null mice which are viable and do not exhibit any obvious
phenotype when kept under normal laboratory confinement and diet, experience serious
difficulties during fasting, a situation that normally results in an enhanced fatty acid
mobilization and increased β-oxidation in the liver as fatty acids represent the major
energy source.
• Confronted to such a metabolic challenge, PPARα null mice are not capable of enhanced
fatty oxidation and rapidly suffer from hypoketonemia, hypothermia, and hypoglycemia.
• Develop obesity with age.
PPAR and trigliceride metabolism
- PPARα lowers trigliceride levels as a result of:
- enhanced lipolysis,
- induction of FA uptake and catabolism
- reduced FA synthesis and VLDL production by the liver
- increased removal of LDL by modifying LDL composition, which increase the affinity of
LDL for LDL receptor.
- PPARγ may decrease levels of triglicerides by increasing lipolysis and clearance of trigliceriderich lipoproteins in adipose tissue.
PPARα and lipid metabolism
• In the liver, PPARα targets form a comprehensive ensemble of genes which participates
in many if not all aspects of lipid catabolism. It includes:
* transport of fatty acids in the circulation,
* their uptake by the hepatocytes,
* intracellular binding by fatty acid binding proteins,
* activation by the acyl-CoA synthase,
* β-oxidation in the peroxisome and mitochondria,
* ω-oxidation in the microsomes.
PPARα and inflammation and wound healing
• Based on PPARα knockout mouse experiments, it seems that PPARα participates in
the control of the inflammatory response:
* it decreases the extent of inflammation possibly via stimulation of catabolism of the
proinflammatory lipid mediators.
* PPARα activation results in the repression of NFκB signaling which leads to
decreased production of proinflammatory cytokines in different cell-types.
• PPARα plays also an important role in the development of skin barrier and is necessary
for the normal healing of skin wound. Its activation is able to:
* counteract epidermal hyperproliferation,
* promote epidermal differentiation,
* correct the cutaneous pathology,
• It suggest that PPARα ligands could be used effectively as therapeutics to treat a variety
of skin diseases.
PPARα
- PPARα expression is highly upregulated under fasting conditions
- Etanol inhibits PPARα activity – it may play a role in development of alcoholic fatty
liver.
alcoholic fatty liver
PPARα mode of action
PPARα increases the entry of FA into the hepatocyte and favors their activation, intracellular transport and catabolism via the
β-oxidation cycle thus diminishing FA pool and TG production. PPARα also increases ketone body (KB) synthesis and TG
clearance by modulating the expression of genes implicated in TG lipolysis. In addition, PPARα influences HDL production in
the hepatocyte by upregulating ApoA-I and apoA-II gene expression. PPARα further controls the reverse cholesterol
transport by increasing cholesterol efflux from macrophages. The net effect on bile acid (BA) synthesis by the liver is less
clear. Finally, PPARα also acts on cholesterol absorption in the intestine.
EC: esterified cholesterol; FC: free cholesterol; CoA: coenzyme A; 12α-H: 12α -hydroxylase.
Lipoprotein Classes and Inflammation
Chylomicrons,
VLDL, and
their catabolic
remnants
> 30 nm
LDL
HDL
20–22 nm
9–15 nm
Potentially proinflammatory
Potentially antiinflammatory
Doi H et al. Circulation 2000;102:670-676; Colome C et al. Atherosclerosis 2000;149:295-302; Cockerill GW et al. Arterioscler
Thromb Vasc Biol 1995;15:1987-1994.
Characteristics of lipoproteins
Eruptive xanthomas
Effect of dyslipidemia
hemorrhage
Atherosclerotic plaques
Effect of dyslipidemia
healthy vessel
~70% lumen reduction
calcification
occlusive plaque
hemorrhage
Atherosclerosis is an Inflammatory Disease
Vessel Lumen
Monocyte
Endothelium
Cytokines
Growth Factors
Metalloproteinases
Cell Proliferation
Matrix Degradation
Foam Cell
Ross R. N Engl J Med 1999;340:115-126.
Macrophage
Intima
Role of LDL in Inflammation
LDL Readily Enter the Artery Wall Where They May be Modified
Vessel Lumen
LDL
Endothelium
Oxidation of Lipids
and ApoB
Aggregation
LDL
Hydrolysis of Phosphatidylcholine
to Lysophosphatidylcholine
Other Chemical Modifications
Modified LDL
Modified LDL are Proinflammatory
Steinberg D et al. N Engl J Med 1989;320:915-924.
Intima
Modified LDL stimulate expression of MCP-1 in endothelial cells
Vessel Lumen
Monocyte
LDL
MCP-1
Endothelium
LDL
Modified LDL
Intima
Navab M et al. J Clin Invest 1991;88:2039-2046.
Differentiation of monocytes into macrophages
Vessel Lumen
Monocyte
LDL
MCP-1
Endothelium
LDL
Intima
Modified LDL
Macrophage
Steinberg D et al. N Engl J Med 1989;320:915-924.
Modified LDL Promote
Differentiation of
Monocytes into
Macrophages
Macrophages express receptors that take up modified LDL
Vessel Lumen
Monocyte
LDL
Adhesion
Molecules
MCP-1
Endothelium
LDL
Modified LDL
Taken up by
Macrophage
Foam Cell
Macrophage
Steinberg D et al. N Engl J Med 1989;320:915-924.
Intima
Modified LDL induces macrophages to release cytokines that
stimulate adhesion molecule expression in endothelial cells
Vessel Lumen
Monocyte
LDL
Adhesion
Molecules
MCP-1
Cytokines
Endothelium
LDL
Modified LDL
Macrophage
Nathan CF. J Clin Invest 1987;79:319-326.
Intima
HDL prevent formation of foam cells, reduced adhesion
molecules and inhibit the oxidative modification of LDL
Vessel Lumen
Monocyte
LDL
Adhesion
Molecules
MCP-1
Endothelium
LDL
Modified LDL
Cytokines
Macrophage
Foam
Cell
HDL Promote Cholesterol Efflux
Mackness MI et al. Biochem J 1993;294:829-834.
HDL Inhibit
Oxidation
of LDL
Intima
MI – myocardial ubfarction, CVA - cerebrovascular accident (stroke), CHD – cardiovascular diseases
Probability of myocardial infarction during 10 years
PPARβ (PPARδ)
•
PPARβ is relatively poorly characterized.
• Its expression is rather ubiquitous, with varying
levels in different organs.
• Endogenous ligand for PPARδ is prostacyclin
(PGI2).
• PPARδ seems to play a very important role in
implantation of embryo.
• It was
maturation.
also
implicated
in
oligodendrocyte
The phases of cutaneous wound healing
• Immediately following cutaneous injury, blood elements and vasoactive amines
extravasate from locally damaged blood vessels within the dermis. Vascular permeability
is temporarily increased to allow neutrophils (PMNs), platelets and plasma proteins to
infiltrate the wound. Vasoconstriction follows, in response to factors released by these
cells.
The phases of cutaneous wound healing
• Coagulation then occurs as platelets aggregate with fibrin, which is deposited in the
wound following its conversion from fibrinogen.
The phases of cutaneous wound healing
• Platelets release several factors, including platelet-derived growth factor (PDGF) and
transforming growth factor β (TGF-β), which attract PMNs to the wound, signalling the
beginning of inflammation.
The phases of cutaneous wound healing
• After 48 h, macrophages replace PMNs as the principal inflammatory cell. Together,
PMNs and macrophages remove debris from the wound, release growth factors, and begin
to reorganise the extracellular matrix.
The phases of cutaneous wound healing
• The proliferation phase begins at about 72 h as fibroblasts, recruited to the wound by
growth factors released by inflammatory cells, begin to synthesise collagen.
The phases of cutaneous wound healing
• Although the rate of collagen synthesis slows down after about three weeks, collagen
crosslinking and reorganisation occur for months after injury in the remodelling phase of
repair
PPAR expression in epidermis during wound healing
PPARβ – wound healing
• Recent studies have demonstrated an involvement of PPARδ in regulation of wound
healing. Its activation:
* contributes to lipid biosynthesis in sebocytes and keratinocytes
* ameliorates inflammatory responses in the skin.
* diminishes proliferation and accelerates differentiation of keratinocytes
* enhances keratinocyte resistance to apoptotic signals.
•
Increased proliferation and death of keratinocytes at the
edges of epidermal wounds in PPARδ mutant mice
most likely participate in the healing delay observed
in these animals.
Effect of PPARβ deficiency on keratinocyte adhesion...
...and in vitro wound healing.
PPARβ − atherosclerosis
• Some studies indicate, that activation of PPARδ may influence atherogenesis, although
the final output of its action is not known yet.
• PPARδ artificial, selective ligand was reported to cause a dramatic rise in HDL
cholesterol, while lowering the levels of LDL small dense lipoprotein, fasting triglicerides,
and insulin.
• On the other hand, PPARδ, whose expression is increased during differentiation of
macrophages, increases the expression of genes involved in lipid uptake and storage, what
may promote the macrophage lipid accumulation and foam cell formation.
PPARβ overexpressing mice
- Overexpression of PPARβ in adipose tissue specifically induces expression of genes
required for fatty acid oxidation and energy dissipation, which then leads to improved lipid
profiles and reduced adiposity.
- Importantly, these animals are completely resistant to obesity that is induced by a highfat diet and by genetic predisposition.
- As predicted, treatment of obese mice with a synthetic PPARβ agonist depletes lipid
accumulation. In parallel, PPARβ-deficient mice challenged with a high-fat diet show
reduced energy and are prone to obesity. Maybe PPARβ serves as a widespread regulator
of fat burning and is a potential target in the treatment of obesity.
- The Marathon Mice are capable of continuous running
of up to twice the distance of a wild-type littermate. This is
achieved by targeted expression of an activated form of
PPARβ in skeletal muscle, which resulted in a dramatic
increase in "nonfatiguing" type I muscle fibers.
marathon mouse
Increased Oxidative Type I Fibers in the PPARδ Transgenic M
Skeletal muscle fibers are generally classified as type I or type II fibers.
Type I fibers (oxidative/slow) are mitochondria rich and mainly use oxidative metabolism
for energy production, which provides a stable and long-lasting supply of ATP, and thus
are fatigue-resistant.
Type II (glycolytic/fast) fibers comprise three subtypes, IIa, IIx, and IIb. Type IIb fibers
have the lowest levels of mitochondrial content and oxidative enzymes, rely on glycolytic
metabolism as a major energy source, and are susceptible to fatigue, while the oxidative
and contraction functions of type IIa and IIx lie between type I and IIb
Metachromatic staining of the plantaris muscle. Type I fibers are stained dark
blue.
Wang et al. 2004
Sceletal muscles in the PPARδ
Transgenic Mice
Muscles in transgenic mice (TG)
are redder than those in wild-type mice
(WT)
Wang et al. 2004
Effect of PPARδ overexpression on obesity
Body weight
Adipose tissue morphology
Wang et al. 2004
Effect of pharmacological
activation of PPARδ on obesity
Old mice fed a high-fat diet
Body weight
Blood glucose level
after glucose injection
Wang et al. 2004
Thank you and see you next week...
What would be profitable to remember in June:
- Expression pattern of PPARα and PPARβ
- Effects of PPARα ligands on dyslipidemia and atherogenesis
- Physiological role of PPARβ
Slides can be found in the library and at the
Heme Oxygenase Fan Club page:
https://biotka.mol.uj.edu.pl/~hemeoxygenase