Peroxisomes are membrane-linked cytoplasmic organelles (with a fine granular matrix) that are found in most animal cells. Chemically they have certain oxidase and catalase enzymes. The oxidase present in them reduces oxygen to hydrogen peroxide with the concomitant oxidation of an appropriate substrate and the catalase present in them reduces the hydrogen peroxide to water either by a perox idatic or catalytic mechanism. That’s why they were given the name “Peroxisomes”. They perform diverse metabolic functions, including H2O2-derived respiration, β-oxidation of fatty acids, and cholesterol metabolism.
Peroxisome proliferators (PPs) are a large class of structurally dissimilar industrial and pharmaceutical chemicals that were originally identified as inducers of both the size and the number of peroxisomes in rat and mouse livers or hepatocytes in vitro after exposure.
The rapid and coordinate induction of liver cell hyperplasia, peroxisome proliferation, and increases in lipid-metabolizing enzymes by PPs gave an early indication that a receptor-mediated mechanism was involved. A member of the nuclear receptor super family that was transcriptionally activated by PPs was cloned from mouse liver in 1990 and was named the peroxisome proliferator-activated receptor (PPAR). Like other members of the vertebrate steroid nuclear receptor super family, PPARs exist in distinct isoforms encoded by separate genes. There are three known PPAR isoforms: PPARα, PPARδ (also known as NUC1 and PPARβ), and PPARγ.
PPAR isoforms perform different physiological functions, based on their divergent patterns of tissue-specific expression, different ligand-binding specificities, and divergent physiological consequences when activated. PPARα is highly expressed in brown adipose tissue and liver, then kidney, heart, skeletal muscle and intestine. PPARβ/δ is ubiquitously expressed, but the highest expression is in the gut, kidney and heart. PPARγ exists in two distinct isoforms, designated PPARγ1 and PPARγ2, that have different tissue distributions and functions. PPARγ1 is found in liver and to a lesser extent in colon, retina, immune system and adipose tissue. PPARγ2 is expressed exclusively in adipose tissue and is a potent regulator of adipocyte differentiation.
Recent evidence demonstrates that PPARs modulate gene expression in a manner similar to that of other nuclear receptors. First, they bind a specific element in the promoter region of target genes. PPAR and some other nuclear hormone receptors bind the promoter only as a heterodimer with the receptors for retinoic acid, RXR (retinoic X receptors). Second, they activate transcription in response to binding of the ligand. For the PPAR:RXR heterodimer, binding of the ligand of either receptor can activate the complex, but binding of both ligands simultaneously is more potent.
PPAR function at cellular level
Much of the function of PPAR can be extrapolated from the identity of their target genes, which so far all belong to path ways of lipid transport and metabolism. PPARα has mostly been a studied in the context of lever parenchymal cells, where it is expressed. The target genes of PPARα are a relatively homogenous group of genes that participate in aspects of lipid catabolism such as, fatty acid uptake through membranes, fatty acid binding in cells, fatty acid oxidation (in microsomes, peroxisomes and mitochondria) and lipoprotein assembly and transport.
Where as PPARα operates in the catabolism of fatty acids in the lever, PPARγ influences the storage of fatty acids in the adipose tissue. With
the C/EBP transcription factor, PPARγ is part of the adipocytes differentiation programme that induces the maturation of pre-adipocytes into fat cells. Most of the PPARγ target genes in a adipose tissue are directly implicated in lipogenic pathways, including lipo protein lipase (LPL), adipocyte fatty acid binding protein (A-FABP or aP2), acyl-CoA synthase and transport protein (FATP).
PPARβ has received little attention, probably because of the lack of connection with important clinical manifestations. However, PPARβ is linked to colon cancer, among other functions, PPARβ regulates the expression of acyl-CoA synthetase in the brain, linking PPARβ to basic lipid metabolism. Moreover, it probably participates in embryo implantation and decidualisation. These data will spur new interest in the study of PPARβ function.
PPARs in whole body physiology
The role of these transcription factors in whole body human physiology and metabolism can best be illustrated by comparing two opposite nutritional states: early absorptive period or fed state and late post-absorptive period or fasting state. In the fed state which in humans is pup to 4 hours after a large meal, carbohydrates and fat enter the circulation in the form of glucose and chylomicrons, respectively. Most glucose is taken up by the lever and if glycogen stores are already filled, used for lipogenesis. The amount of the transcription factor sterol response element binding protein-1 (SREBP1) rises in the fed state, which promotes the glycolytic conversion of glucose into acetyl-CoA and subsequently the synthesis of fatty acids from acetyl-CoA. Fatty acids are converted to triglycerides and packaged into very low density lipoproteins(VLDL).
In adipose tissue, the amounts of SREBP and PPARγ are elevated, probably because of regulation by insulin. PPARγ is a direct target gene of SREBP, which emphasizes the cooperative and additive functions between these two types of receptor. In addition, SREBP1 may be involved in producing an endogenous ligand (probably fatty acids) for PPARγ. The overall effect is stimulation of the uptake of glucose and fatty acids and their subsequent conversion to triglycerides.
Triglyceride storage cause increases production of the hormone leptin in the adipose tissue. Leptin is the protein product of the ob gene, whose deletion leads to severe obesity in mice. Its expression is increased by long term overfeeding as part of a feedback mechanism to limit further food intake and weight gain. Consistent with the role of PPARγ in promoting lipogenesis, production of leptin in adipose tissue is under negative control by PPARγ. Paradoxically, expression of both PPARγ and leptin is reduced by fating and increased by feeding. In the latter case, PPARγ may attenuate the increase in leptin expression to limit wasteful lipolysis and fatty acid oxidation processes which are stimulated by leptin. If the above scenario is correct, decreased PPARγ expression may lead to increased leptin levels and, as a result, to lower food intake and weight gain. Studies with PPARγ+/- mice indicate that this is
the case. A different situation exists in the late post-absorptive or fasting state. In the lever, fatty acids are oxidized to acetyl–CoA and subsequently to ketone bodies, such as acetoacetate and β-hydroxy butyrate. Both processes are strongly stimulated by PPARα expression of which is elevated upon fasting. Fatty acids are ligands for PPARs, so it is possible that the large amount of fatty acids liberated from the adipose tissue can stimulate their own metabolism by activating PPARα. Experiments with PPARα null mice show that PPARα is important in the hepatic response to fasting. When fasting these mice suffer from a defect in fatty acid oxidation and ketogenesis, resulting in elevated plasma free fatty acids, hypoketonaemia, hypothermia and hypoglycaemia. The hypoglycaemia emphasizes the important interplay between fatty acid and glucose metabolism in energy homeostasis.
Therapeutic significance of PPAR ligands
Emerging evidence indicates that PPs have the ability to suppress the growth of different types of human cancer. Early indications that PPs can suppress growth of tumours came from traditional studies of the growth promotion effects of PPs in the livers of rats. Although PPs promote the growth of basophilic lesions, there was a paradoxical suppression of both c-glutamyl transpeptidase-positive and ATPase-deficient foci, indicating that PPs through PPARs may inhibit growth in these lesions.
A large number of studies have demonstrated growth inhibition properties of PPARα and PPARγ ligands on human tumour cell lines. A large number of tumour types appear to be sensitive to PPs, including cells from prostate cancer, monocytic leukemia, ovarian carcinoma, hepatoma, liposarcoma, and breast cancer. Growth inhibition of these cell lines occurred through a number of distinct mechanisms, including increases in necrosis, apoptosis, and growth arrest. In addition, increases in differentiation to a cell type expressing markers of adipocyte phenotype also occurred in liposarcoma and breast cancer cell lines. Increases in PPAR expression do not seem to be required for growth inhibition, as PPARα
was not altered in hepatoma cells and PPARγ was unchanged in monocytic leukemia and prostate cancer cells. In all cases examined, however, PPARs were expressed to varying degrees in the target tissues .
PPAR
ligands have potent anticancer activity in vivo. In studies using immunodeficient mice injected with human prostate or human breast cancer cells, treatment of mice with the PPARγ ligand troglitazone decreased tumour volume and weight. It is interesting to note that treatment with a combination of
troglitazone and all-trans-retinoic acid was more potent at inhibition of tumour growth in these two studies. Treatment with dehydroepiandrosterone, a primary steroid precursor and a PP, decreased the number of ethylnitrosourea-induced rat mammary tumours. Lastly, the intermediate-to-high-grade liposarcomas in patients treated with a thiazolidinedione exhibited extensive lipid accumulation and up regulation of genes involved in terminal adipocyte differentiation, as well as down-regulation of a marker of cell proliferation. In summary, many different types of cancer may respond favourably to PP therapy, providing a rational basis for anticancer medicines that work through PPAR family members.
There is conflicting evidence of the effects of PP treatment on colon cancer cell growth. Similar to the therapeutic effects of PPs, discussed above, treatment of human colon cancer cell lines with troglitazone resulted in decreases in cell replication, in G1 cell-cycle arrest, and in increases in expression of markers of enterocyte cell differentiation. Consistent with the therapeutic effects of
PPs, mutations in PPARγ were found in human colon cancers that resulted in a decreased ability of PPARγ to be activated by ligands. These data indicate that
a functional PPARγ is required for normal growth properties of human colon cells. In contrast, treatment of Min mice predisposed to intestinal neoplasia with the PPARγ ligands troglitazone or BRL-49,653 resulted in increases in the number of colon tumours but not small-intestine tumours.
Increases in the protein b-catenin, which has been linked to colon cancer, were observed in the colon in these mice after BRL-49,653 treatment, pointing to a reprogramming of gene expression important in tumourigenesis. Thus, the promise of using thiazolidinediones to treat colon cancer is overshadowed by the possibility that these compounds may actually increase susceptibility of colon cancer in certain human subpopulations. Further work on the significance of findings in the Min mice is needed.
PPARs and inflammation
There is increasing evidence that PPARs are capable of inhibiting inflammatory responses in certain cell types. This effect may be mediated by at least two mechanisms. First, proinflammatory lipid metabolites may serve as ligands for PPARs, thereby activating PPAR-responsive enzymes responsible for their clearance. This has been demonstrated for PPARα-mediated catabolism of LTB4, and PPARc-induced 12/15-lipoxygenase catabolism of linoleic acid and arachidonic acid to PPARc ligands. It is interesting to note that interleukin (IL)-4 upregulates both
12/15-lipoxygenase and PPARc, which suggests a new paradigm for the regulation of nuclear receptor function by cytokines. Second, PPARs may influence cytokine
induction by other transcription factors with roles in mediating inflammation, such as signal transducers and activators of transcription, NF-κB, and activator
protein-1. This mechanism appears to be operative via PPARc, as well as PPARα. For example, activation of PPARγ inhibits transcription of inducible nitric oxide synthase, gelatinase B, and scavenger receptor A in activated macrophages, and alpha tumour necrosis factor, IL-1b, and IL-6 in monocytes, by a mechanism that occurs in the absence of PPARγ-DNA interaction. In a similar fashion, activation of PPARα in activated aortic smooth muscle cells leads to decreased expression of IL-6 and cyclooxygenase-2 (COX-2). Possible explanations for these effects include PPAR titration of essential transcriptional cofactors, such as CBP/p300 and SRC-1, used by other transcription factors, or perhaps by direct inactivation by PPAR-transcription factor interaction.
A direct role for PPARα in down-regulating inflammatory responses in vivo is now well established. Treatment of wild-type but not PPARα-null mice with diverse PPs resulted in down-regulation of a number of acute-phase response genes normally induced in the liver after a localized inflammatory stimulus. In PPARα-null mice, acute-phase response genes are expressed at higher levels than in wild-type mice LTB4- and arachidonic acid-induced ear swelling is prolonged, and there are higher levels of age-associated NF-κB activity and regulated genes,
including IL-6, IL-12, COX-2, and tumour necrosis factor alpha (TNFα). Similar responses may occur in humans because patients receiving therapeutic doses of
hypolipidemic drugs have decreased acute-phase proteins and cytokine levels in serum. These data indicate that the PPAR family members may be attractive
candidates for therapeutic intervention in chronic inflammatory diseases often associated with aging.
Conclusion
In the decade since the first PPAR was cloned, a substantial body of research has revealed that PPARs exist in three isoforms with distinct, sometimes overlapping, roles in regulating fatty acid metabolism, glucose homeostasis, cell growth and differentiation, and inflammation. Although much has been accomplished, many important questions remain. A large number of both endogenous
and exogenous PPAR ligands have been identified. It is likely that additional natural ligands, and the conditions under which they are produced, will be uncovered. Considerable effort has already been directed toward understanding the complex interactions between PPARs, other nuclear receptors, and receptor cofactors. Forthcoming research will address how the architecture of these complexes determines tissue specific patterns of gene expression. This, in turn, should facilitate identification of additional genes that are both directly and indirectly regulated by the different PPARs. Clinically, PPs are already emerging as important mediators in the progression of certain chronic human diseases. Thus, it will be important to explore how these receptors can be manipulated therapeutically to delay disease progression or alleviate symptoms. Finally, given that several PPs are potent rodent carcinogens, another significant challenge will be to gain a more complete understanding of how accurately rodent cells predict human responses. If they are not good surrogates, it will be important to understand why. Answers to these questions will greatly enhance our ability to more accurately estimate the true relative
risk of adverse health effects in humans receiving chronic therapeutic or environmental exposure to various PPs. Looking toward the future, research on the mechanisms of PPAR activation promises to continue to yield exciting and biomedically beneficial information.
(The author is with Dept of Pharma Management, Manipal College of Pharmaceutical Sciences, Manipal)