FYI

[Guest guest]

To All,

FYI, this article is dry and technical, however I included it to

demonstrate that scientists are now at the gene level with understanding and

treatment. It may even have implications for Lyme disease.

Larry NV

Clinical Significance of Peroxisome Proliferator-Activated Receptors in

Health and Disease

M. Loviscach, MD, and R.R. Henry, MD

Department of Medicine

University of California, San Diego

La Jolla, Calif

VA San Diego Healthcare System

San Diego, Calif

[Medscape Diabetes & Endocrinology, 1999. © 1999 Medscape, Inc.]

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Introduction

Peroxisome proliferator-activated receptors (PPARs) are members of the

nuclear hormone receptor family of transcription factors, a diverse group of

proteins that mediate ligand-dependent transcriptional activation and

repression.[1] They modulate DNA transcription by binding to specific

peroxisome proliferator-response elements (PPREs) on target genes.

Interest in PPARs increased dramatically after their existence was first

described in mammals and humans. Research has shown that PPARs are involved

in the regulation of lipid and glucose metabolism, adipocyte

differentiation, inflammatory responses, and cancer. Therefore,

pharmacologic agents that target PPARs may have a wide range of therapeutic

applications, and could potentially provide medical interventions for

diseases as diverse as cancer and coronary heart disease. However, as

promising as most of the current data may be, most is preliminary in nature

and awaits confirmation by additional basic and clinical research.

This review will provide evidence for the involvement of the different forms

of PPARs in a variety of metabolic disorders, inflammation-driven diseases,

and cancer. A brief discussion of the factual or hypothetical clinical

implications of the respective findings will also be presented.

Expert Column - Clinical Significance of Peroxisome Proliferator-Activated

Receptors in Health and Disease continued...

[Medscape Diabetes & Endocrinology, 1999. © 1999 Medscape, Inc.]

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Tissue Distribution, Ligand Specificity, and Forms of PPARs

Three forms of PPARs have been described to date: PPAR-alpha, PPAR-beta

(also designated PPAR-delta, NUC1, and FAAR), and PPAR-gamma. Although it

appears that the formation of a PPAR and retinoid X receptor (RXR)

heterodimer is required for PPRE binding and activation of gene

transcription,[2] certain PPAR-alpha mutants that are deficient in their

ability to heterodimerize with RXRs retain their ability for functional PPRE

interaction.[3] This suggests either that there are additional

heterodimerization partners besides RXR or that other nuclear proteins might

compensate for the decreased binding of RXRs to these mutants. A basic model

of PPAR function is presented in Figure 1.

Figure 1. (click image to zoom) Basic model of PPAR function

PPAR-alpha

In humans, various tissues express PPAR-alpha, including skeletal muscle,

liver, kidney, and vascular endothelial cells.[4] Studies have shown that

PPAR-alpha is involved in the control of lipoprotein metabolism,[5,6] fatty

acid oxidation,[7,8] and the cellular uptake of fatty acids.[9] In addition

to being an established target for the treatment of dyslipidemia and a

possible contributory factor in the pathogenesis of lipid disorders,

PPAR-alpha has been implicated in the regulation of inflammatory responses

in vascular endothelial cells and atherosclerosis.[10,11] The

transcriptional activity of PPAR-alpha is stimulated by insulin,[12]

fibrates, phenylacetate and its analogues,[13] the selective agonist

WY14643,[14] JTT-501,[15] GW2331,[16] and PD72953.[17]

PPAR-beta

The existence of PPAR-beta protein has been demonstrated in a number of

different tissues in the adult rat.[18] In animal models, this receptor

appears to have a role in oligodendrocyte differentiation[19] and

spermatogenesis.[20] Although it has been detected in human tissues,[21] its

distribution, regulation, and functions in humans remain to be determined.

PPAR-gamma

The third and probably the best characterized receptor, PPAR-gamma, is known

to play a critical role in adipocyte differentiation and fat deposition and

is highly expressed in this tissue.[21-23] In addition, both PPAR-gamma

mRNA[22,23] and PPAR-gamma protein[24] have been detected in human skeletal

and cardiac muscle.[23,24] The mRNA for PPAR-gamma has also been found in

liver,[23] kidney, small intestine,[25] bladder, and spleen.[26] The

PPAR-gamma mRNA exists as 3 isoforms, gamma1, gamma2, and gamma3, each

transcribed from its own promoter.[27] PPAR protein, however, exists only as

2 discernible isoforms, gamma1 and gamma2, the products of alternative

splicing. The protein translated from PPAR-gamma3 mRNA is indistinguishable

from PPAR-gamma1 protein.[27] Compared with the gamma1 isoform, PPAR-gamma2

contains an N-terminal 30 amino-acid extension and displays a lower mobility

on SDS-PAGE gel.[28] Activators of PPAR-gamma include the natural

prostaglandin (PG) derivative 15-deoxy-delta 12,14-PGJ2,[29] the synthetic

insulin-sensitizing thiazolidinediones (TZDs) troglitazone (Rezulin®),

pioglitazone (Actos®), and rosiglitazone (Avandia®),[30-34] GW1929,[35]

JTT-501,[15] PD72953,[17] and indomethacin and other nonsteroidal

anti-inflammatory drugs (NSAIDs).[36]

PPAR Ligands May Have Diverse Clinical Applications

The intense clinical interest in the role of PPARs in health and disease was

sparked by the discovery that PPAR-gamma mediates the antidiabetic and

adipogenic actions of the TZDs.[30,32] Recent attention has focused on the

TZD troglitazone because its use has been associated with the rare

idiosyncratic development of potentially lethal hepatotoxicity. Although at

present no similar side effects have been observed with either rosiglitazone

or pioglitazone, the Food and Drug Administration has issued guidelines for

periodic liver testing for patients undergoing treatment with all of these

agents. However, because evidence to date does not indicate that liver

toxicity is attributable to TZDs as a class or to PPAR-gamma agonists in

general, they will not be discussed further in this review.

With our expanding knowledge of PPAR biology came the realization that drugs

acting through PPARs had potential clinical applications beyond glucose,

lipid, and fat metabolism. Activation of PPAR-alpha through the aromatic

fatty acid phenylacetate produced cytostatic effects on human prostate

carcinoma, melanoma, and glioblastoma cell lines,[13] thus stimulating

research into the role of PPARs in treating other diseases such as those

resulting from underlying chronic inflammatory processes.

The Role of PPAR-gamma in Glucose Metabolism

In order to illustrate the clinical importance of PPARs, we will begin by

considering their role in glucose metabolism. The antidiabetic TZDs, the

most well-known PPAR ligands, are highly selective for PPAR-gamma, having

minimal activity toward PPAR-alpha and PPAR-beta.[37] The TZDs produce their

antihyperglycemic effect in type 2 diabetic patients by selectively

enhancing or partially mimicking certain actions of insulin and thereby

increasing insulin-dependent glucose disposal and reducing hepatic glucose

output.[38] In addition, TZDs have lipid-lowering activity that may offer

potential benefit in reducing morbidity and mortality associated with

diabetes-related cardiovascular complications.[39] More recent studies

performed in rodents suggest that PPAR-gamma agonists protect against islet

cell degeneration and preserve the physiologic first- and second-phase

insulin secretion pattern.[40-42]

Besides the antidiabetic TZDs, a number of other structurally unrelated

PPAR-gamma ligands have been identified with glucose and, in some cases,

lipid-lowering properties. These compounds include conjugated linoleic

acid,[43] as well as derivatives of N-(2-benzoylphenyl)-L-tyrosine,[44,45]

isoxazolidinedione (ie, JTT-501, a mixed PPAR-alpha and gamma agonist also

promising distinct lipid-lowering traits),[46] and phenylacetic acid (a

mixed PPAR-beta (delta) and PPAR-gamma agonist).[47] The diversity in

structure of these similarly active PPAR-gamma ligands further supports the

idea that activation of nuclear PPARs is responsible for the observed

insulin-sensitizing and glucose- and lipid-lowering effects.

Consistent with the observed antidiabetic properties of PPAR-gamma agonists,

both its mRNA and receptor protein are expressed in tissues involved in

glucose homeostasis in humans, namely skeletal muscle,[22-24,48] adipose

tissue,[21-23,26] and liver.[23,25] In human skeletal muscle, which is the

major site of impaired insulin action in type 2 diabetes and obesity,

PPAR-gamma mRNA expression can be acutely regulated by insulin.[48]

Incubation of primary human skeletal muscle cell cultures with the TZD

troglitazone increases the expression of PPAR-gamma mRNA and the content of

PPAR-gamma protein, suggesting that PPAR ligands can stimulate the

expression of their own receptors.[49]

PPAR-gamma Is Highly Expressed in Adipose Tissue

Human adipose tissue highly expresses PPAR-gamma mRNA, and it has been

suggested that the antidiabetic effects of the TZDs are mediated through

their action on adipose tissue. However, in hyperlipidemic, hyperglycemic,

and hyperinsulinemic mice totally devoid of white and brown adipose tissue,

treatment with the PPAR-gamma agonist troglitazone normalized glucose

tolerance and significantly decreased insulin levels.[50] These results

strongly suggest that the antidiabetic effect of PPAR-gamma agonists is not

solely mediated by the activation of this nuclear receptor in adipose

tissue.

Nevertheless, some investigators support the idea that PPAR-gamma agonists

exert their effects principally or even solely on adipocytes, which in turn

modulate metabolic responses in muscle. This conclusion is derived from

observations that the amount of PPAR-gamma mRNA detected in muscle equals 5%

or less of the PPAR-gamma mRNA expressed in adipose tissue, and that the

treatment of some cell lines with certain PPAR-gamma ligands results in

their differentiation into adipocytes.[51] However, the level of mRNA

expression does not always directly correlate with the tissue content of the

physiologically active receptor protein. Experiments conducted in our

laboratory indicate that the amount of PPAR-gamma protein in human muscle

biopsies averaged 70% of the amount of PPAR-gamma protein detected in

adipose tissue biopsies from the same individuals (Loviscach et al, 1999,

submitted for publication). Furthermore, not all cells differentiate into

adipocytes when treated with PPAR-gamma ligands, and not all PPAR-gamma

ligands lead to adipocyte differentiation.

It was recently demonstrated that high-affinity ligands of PPAR-gamma

inhibit the differentiation of 3T3-L1 preadipocytes,[52] a cell line with

inherent adipogenic potential. Therefore the adipogenic property of

PPAR-gamma ligands seems to depend on the tissue type and the availability

of certain cofactors (such as PGC-2, a receptor isoform-selective cofactor

of PPAR-gamma) that mediate its adipogenic effects.[53]

PPARs Regulate Fatty Acid Metabolism

Based on observations that hypolipidemic drugs that activate PPARs inhibit

fatty acid synthesis and stimulate the peroxisomal beta-oxidation pathway,

PPARs were believed to play an important role in the regulation of fatty

acid metabolism[54] and possibly in the development of obesity. This

hypothesis was further supported by the detection of a PPRE in the promoter

region of the gene for malic enzyme, which is involved in lipogenesis.[55]

The important role of PPARs in the development of lipid disorders was

underscored by the observation that in human apolipoprotein A-1 (apo A-1)

transgenic mice, PPARs mediate the ability of fibrates (ie, fenofibrate,

gemfibrozil) to increase liver apo A-1 production and, as a result, increase

high-density lipoprotein (HDL) cholesterol.[56,57] In addition, the

hypotriglyceridemic effects of both fibrates and TZDs are mediated through a

PPAR-mediated increase in the transcription of the lipoprotein lipase

gene.[58] This effect of the TZDs was predominantly mediated by activation

of PPAR-gamma in adipocytes, while fibrates activated PPAR-alpha in the

liver.

It is important to mention that rodents and humans show a distinctly

different response to treatment with fibrates. In rodents, fibrates produce

a PPAR-alpha-mediated decrease in HDL as a result of a decrease in

transcription of apo A-1 in liver. In man, fibrates increase plasma levels

of HDL via an induction of human apo A-1 gene expression. This contrary

effect of fibrates on mice and men is attributed to sequence differences in

regulatory elements in their respective gene[5] and possibly is also

responsible for the lack of any undesirable peroxisomal proliferation and

tumor genesis in human livers.

Do Mutations in PPAR-gamma Affect Glucose Metabolism?

The emerging physiologic role of PPAR-gamma in diabetes and lipid disorders

prompted further investigation into whether mutations of the PPAR-gamma gene

are associated with the development of type 2 diabetes, obesity, and

hyperlipidemia. The human PPAR-gamma gene is localized on chromosome 3, band

3p25.[59] Screening for mutations in the coding region of the PPAR-gamma2

gene led to the discovery that a single amino-acid missense mutation

(Pro12Ala) decreased the ability of the receptor to stimulate gene

transcription.[40,60] This genetic variant was not associated with the

occurrence of diabetes, obesity, or hyperlipidemia in type 1 or type 2

diabetic subjects.[40,60-62] However, a subgroup of severely obese patients

(BMI >30 kg/m2) who had the Pro12Ala mutation exhibited increased insulin

sensitivity.[61]

PPAR-gamma2 plays a key role in adipocyte differentiation, and therefore

mutations of the gene for this factor may predispose individuals to obesity.

A Pro115Gln mutation in PPAR-gamma2 that accelerates the differentiation of

adipocytes may cause obesity,[63] and a Pro12Ala variant was associated with

obesity in white populations.[64,65] These observations do not currently

support a major role for PPAR-gamma mutations in the etiology of type 2

diabetes, but suggest that a mutation may be involved in the development of

obesity.

PPAR-Targeted Treatment for Metabolic Disorders

In summary, the activation of PPAR-alpha and PPAR-gamma isoforms by diverse

ligands leads to variable reductions of plasma levels of triglycerides,

glucose, and insulin levels as well as an improvement in insulin resistance

(Figure 2). The effect on glucose homeostasis is unlikely to be mediated

solely through PPAR-gamma activation in adipose tissue. Skeletal muscle,

which is the major site of impaired insulin action in type 2 diabetes and

obesity, is likely to be regulated by PPAR activation in an adipose

tissue-independent manner. Ligand activation of PPAR-gamma might also prove

beneficial in the prevention of islet cell degeneration leading to diabetes.

Although there is evidence for the involvement of certain mutations in the

development of obesity, a mutation in the PPAR-gamma gene associated with

diabetes has not yet been found.

Figure 2. (click image to zoom) Effect of PPAR agonists on glucose and

lipid metabolism

The Role of PPARs in Inflammation: Atherosclerosis, Rheumatoid Arthritis,

and Crohn's Disease

Inflammation is a local immune response to " foreign " molecules, infection,

and injury. Research suggests that both PPAR-alpha and PPAR-gamma are

involved in mediating inflammatory processes. Leukotriene B4, an activator

of PPAR-alpha,[66] is a potent chemotactic agent that initiates,

coordinates, sustains, and amplifies the inflammatory response. Mice that

are deficient in PPAR-alpha exhibit a prolonged response to inflammatory

stimuli, also suggesting a modulatory effect of PPAR-alpha on inflammation.

In hyperlipidemic patients, the PPAR-alpha ligand fenofibrate decreases the

plasma concentrations of interleukin (IL)-6, fibrinogen, and C-reactive

protein. In human aortic smooth muscle cells, PPAR-alpha ligands inhibit

IL-1 induced production of IL-6 and prostaglandin as well as the expression

of cyclo-oxygenase-2.[10] Both PPAR-alpha and PPAR-gamma are expressed in

human monocyte-derived macrophages, which participate in inflammation and

atherosclerotic plaque formation. Ligand activation of PPAR-gamma results in

cell death (apoptosis) of unactivated, differentiated macrophages. Both

PPAR-alpha and PPAR-gamma ligands induce apoptosis of macrophages activated

by tumor necrosis factor-alpha (TNF-alpha)/interferon-gamma (IFN-gamma).[11]

Atherosclerosis and Rheumatoid Arthritis

At present, research on the role of PPARs in chronic inflammatory diseases

has focused mainly on atherosclerosis. In atherosclerosis and rheumatoid

arthritis, activated macrophages exert pathogenic effects. A variety of

PPAR-gamma agonists, such as prostanoids, TZDs, and NSAIDs, suppress

monocyte elaboration of inflammatory cytokines.[67] The expression of

PPAR-gamma was found to be markedly increased in activated macrophages, and

stimulation of PPAR-gamma by either locally produced prostaglandin

metabolites or synthetic ligands inhibited the expression of substances that

promote inflammatory tissue destruction.[68] In addition, PPAR-gamma is

expressed in macrophage foam cells of human atherosclerotic lesions[69,70]

and endothelial cells (ECs) of human carotid arteries. Gene expression of

plasminogen activator inhibitor type-1 (PAI-1), a major physiologic

inhibitor of fibrinolysis and a predictor of risk of myocardial infarction

and venous thrombosis, was found to be down-regulated by PPAR-gamma.[71]

In addition to PPAR-gamma, human carotid arteries also express PPAR-alpha.

Adhesion molecule expression on the EC surface is critical for leukocyte

recruitment to the site of atherosclerotic lesions. The TNF-alpha-induced

vascular cell adhesion molecule-1 (VCAM-1) was inhibited in cultured human

ECs by the PPAR-alpha activators fenofibrate and WY14643 in a time- and

concentration-dependent manner. Finally, PPAR-alpha activators significantly

reduced the adhesion of U937 cells to cultured human ECs.[72]

Crohn's Disease

A recently published study on the role of PPAR-gamma in Crohn's disease

suggests an expansion of academic interest in the contribution of PPARs to

other inflammatory diseases. The increase in mesenteric adipose tissue seen

in patients with Crohn's disease was associated with overexpression of

PPAR-gamma and TNF-alpha. The investigators suggest that PPAR-gamma

activation is responsible for this mesenteric adipose hypertrophy.[73]

Although this conclusion needs to be supported by additional data, this

study illustrates the growing appreciation of PPARs as transcription factors

with a variety of tissue- and cofactor-dependent functions.

The Role of PPARs in Cancer

Shortly after their discovery, PPARs were implicated in the development of

certain cancers. The observation that certain PPAR ligands (including

hypolipidemic drugs such as clofibrate, plasticizers, and herbicides) induce

peroxisome proliferation and act as rodent hepatocarcinogens[74] generated

considerable interest. High-fat diets have been linked to an increased risk

of colon, breast, and prostrate cancer, providing further hypothetical

support for a role of PPAR in cancer genesis. Although research performed in

rodents supports a contributory role of PPAR-gamma to the development of

colorectal cancer,[75,76] there is no evidence to suggest that it promotes

tumors in humans. Moreover, it has been shown that the activation and

increased expression of PPAR-alpha induced by clofibrate or the aromatic

fatty acid phenylacetate and its analogues promote tumor cytostasis and

differentiation in human prostate carcinoma, melanoma, and glioblastoma cell

lines.[13] Therefore, in humans, the PPARs may prove beneficial in the

prevention of certain cancers.

PPAR-Induced Terminal Differentiation: A Novel Therapeutic Approach for

Cancer?

It became apparent that the induction of terminal differentiation by PPARs

represents a therapeutic approach to certain human malignancies. High levels

of PPAR-gamma are expressed in different histologic types of human

liposarcoma,[77] certain human colon cancer cell lines,[78] human primary

and metastatic beast adenocarcinomas,[79] human prostate cancer cells,[80]

and a human monocytic leukemia cell line.[81] Treatment with the

PPAR-gamma-specific ligand pioglitazone induced primary human liposarcoma

cells to undergo terminal differentiation.[77] The results of a clinical

study on patients with advanced liposarcoma, who were treated with the

PPAR-gamma-specific ligand troglitazone indicated that lineage-appropriate

differentiation can also be induced in a solid tumor.[82] Ligand activation

of PPAR-gamma induces terminal differentiation of malignant breast

epithelial cells.[79] The combination of a PPAR-gamma ligand (troglitazone)

and a retinoic acid receptor-specific ligand (all-trans retinoic acid)

synergistically and irreversibly inhibited growth in breast cancer cells but

not in normal breast epithelial cells. The same treatment caused significant

cell death and fibrosis in a murine breast tumor model without toxic effects

on the mice.[83] Treatment of a prostate cancer cell line with different

PPAR-gamma ligands produced an antiproliferative effect and dramatic

morphologic changes consistent with decreased malignancy. Short-term culture

of surgically obtained human prostate cancer tumors with troglitazone

produced marked and selective necrosis of the cancer cells but not the

adjacent normal prostate cells.[80] Activation of PPAR-gamma with either BRL

49653 or troglitazone led to growth inhibition and resulted in G1 cell cycle

arrest in a series of 4 and 6 different colon cancer cell lines,

respectively. Interestingly, the degree of growth inhibition correlated with

the level of functional PPAR-gamma.[84,85]

In a recent study, 4 somatic mutations of PPAR-gamma, each greatly impairing

the function of the receptor, have been identified among 55 cases of

sporadic colon cancers. One of these mutations conferred a normal response

to receptor binding of synthetic PPAR-gamma ligands and a decreased binding

affinity for natural ligands.[86] However, although this mutation offers an

explanation for the beneficial effects observed in treating colon cancer

with synthetic PPAR-gamma ligands, it does not seem to be associated with

the majority of colon carcinomas.

PPAR-Targeted Treatment for Cancer

In summary, ligand activation of PPARs expressed in several cultured human

malignomas converts cancer cells into a morphologically and

immunohistochemically less malignant and better differentiated state and

induces growth arrest and apoptosis of malignant cells (Figure 4).

Furthermore, these effects could also be induced in a solid tumor. The PPARs

may provide a promising novel target for prevention and treatment of a range

of malignant tumors in humans.

Figure 4. (click image to zoom) Effect of PPAR agonists on malignant cells

Therapeutic Potential of PPAR-Mediated Therapies

PPARs mediate a variety of processes in glucose and lipid metabolism,

inflammatory responses, and regulation of cellular differentiation and

death. They exist in 3 forms, PPAR-alpha, PPAR-beta and PPAR-gamma. Each

form is expressed in various different tissues and can be activated by a

number of different ligands, most of them being specific for one form of

PPAR. Neither the expression nor overexpression of PPARs has been

convincingly shown to be responsible for the development of the pathologic

changes that are associated with either chronic inflammation or malignant

disease. On the contrary, activation or induction of PPARs has produced

beneficial physiologic effects on human cells in vitro as well as in

patients having diseases as diverse as diabetes and liposarcoma. Although

the data available to date are mostly preliminary and conclusions must be

drawn with caution, it is apparent that the discovery of PPARs opens a new

and exciting chapter in our understanding of human health and disease and

may lead to the development of novel specifically acting drugs in

therapeutic areas other than diabetes and lipid metabolism, where their

principle of function was first revealed.