PPARs and Their Relevance to Clinical Practice

At a symposium held in conjunction with the American Association of Clinical Endocrinologists annual meeting in Chicago, Illinois, three leaders in endocrinology presented the latest research on peroxisome proliferator-activated receptors (PPARs) and how PPAR ligands can prevent the development of diabetes as well as some of the cardiovascular complications associated with diabetes. Topics included the role of PPAR ligands in protecting beta cells, as well as inhibiting the development of atherosclerosis, and restenosis.

This program was supported by unrestricted educational grants from Takeda Pharmaceuticals North America, Inc., and Eli Lilly & Company.

The Role of PPAR Activators in ß-cell Preservation

Reducing the workload of beta cells in the pancreas is fundamental to treating type 2 diabetes, according to Charles F. Burant, MD, PhD, associate professor of Internal Medicine at the University of Michigan Medical School in Ann Arbor, Michigan. Early in the development of type 2 diabetes, patients have increased insulin resistance, but the body compensates by increasing insulin secretion to keep blood glucose normal. Over time, however, the increased demand on the beta cells lead to ß-cell dysfunction and decreased ß-cell mass, which in turn, leads to a relative lack of insulin and
progressive hyperglycemia.

The UK Prospective Diabetes Study has shown that by the time persons are diagnosed with type 2 diabetes their ß-cell function is about 60% of normal. Whether the 40% reduction is the result of cells secreting less insulin or a decrease in b-cell mass is unclear, but likely represents a combination of the two processes.

Intervention with proper diet, exercise and metformin can attenuate the decline in b-cell function and prevent the onset of diabetes if treatment is begun early as demonstrated in the Diabetes Prevention Program (N Engl J Med 2002;346:393–403). Another possible option is to employ the use of the thiazolidinediones (TZDs) to preserve beta cell function. TZDs act as activators of peroxisome proliferator-activated receptors (PPARs) and by using this
treatment “TZDs can reverse the intrinsic dysfunction of the ß-cell and probably preserve ß-cell mass,” stated Dr. Burant.

PPAR Activators and ß-Cell Dysfunction
In a study at the University of Chicago (J Clin Invest 1997;100:530– 537), TDZ was found to increase beta cell secretion rates in patients with IGT as well as improve beta cell responses to oscillating changes in blood glucose, the latter an index of b-cell health. Similar results have been seen in other conditions with insulin resistance such as polycystic ovary disease. The most dramatic results with TZDs in preventing the development of type 2 diabetes was recently shown in a study involving a group of Hispanic women with a history of gestational diabetes, who if left untreated have a 50% risk of developing type 2 diabetes within 5 years. Treatment with TZDs showed that after 30 months, only 19% of the women developed type 2 diabetes compared to 55% in the placebo group (Thomas Buchanan, Diabetes, in press). It was apparent from the study that women whose beta cell function responded better to the TDZ (i.e., showed significant decrease in beta cell workload) did not develop diabetes. In patients that respond well to TZDs by reducing the workload of ß-cells and later taken off the drug, the rate of progression to type 2 diabetes remains low.

PPAR Activators and ß-Cell Dysfunction
In addition to ß-cell dysfunction, many believe that there is a decrease in beta cell mass in type 2 diabetes. Studies in fatty Zucker rats have shown that increased blood glucose is correlated with a time-dependent decrease in ß-cell mass, as assessed by staining for insulin positive cells in the pancreas. If these animals are given a TDZ (rosiglitazone), no decrease in beta cell mass is observed (Diabetes 2001;50:1021–1029). The mechanism of action for TDZ protective effects on beta cell function and mass is unclear but may be due to these drugs preventing apoptosis and perhaps a decline in
neogenesis of ß-cells.

Conclusion
The growing consensus that TZDs can prevent b-cell dysfunction, prevent decline in ß-cell mass, and ultimately, prevent the development of type 2 diabetes requires that patients at high risk for developing type 2 diabetes be treated early. Recent data also indicates that PPAR activators may alter the ratio of beta cell death to ß-cell neogenesis and may be of some use in later stages of type 2 diabetes.

PPARs in Vascular Inflammation

Most type 2 diabetes patients will eventually develop cardiovascular disease. “Type 2 diabetics have a significantly higher risk to develop cardiovascular disease as non-diabetics,” stated Bart Staels, PhD, research group leader in the department of atherosclerosis at the Institut Pasteur de Lille and professor in the faculty of pharmacy at the Université de Lille II in Lille, France. One reason that type 2 diabetes patients develop cardiovascular disease is because they develop atherosclerosis.

PPARs can decrease the progression of atherosclerosis via two mechanisms, one indirectly and one directly. First, PPARs can indirectly reduce atherosclerosis development by correcting some of the metabolic abnormalities associated with the insulin resistance syndrome. Second, PPARs (PPARa, PPARb, and PPARg) are expressed in atherosclerotic lesions of human coronary arteries and have direct anti-inflammatory effects in the different cell types of the vascular wall.

PPARs and the Inflammation Response: In Vitro Evidence
Atherosclerosis is a complex multifactorial process that involves inflammatory and fibroproliferative responses to various stimuli acting on the vascular wall. Oxidized or glycated LDL particles and other pro-atherogenic substances activate endothelial cells resulting in the attraction of leukocytes.These leukocytes secrete cytokines, matrix metalloproteinases, and chemoattractants that trigger the influx of vascular smooth muscle cells, T lymphocytes, and monocytes which differentiate into macrophages. PPARs are expressed in all these cells and modulate the recruitment of leukocytes to endothelial cells, control the inflammation response and lipid homeostasis of monocytes/macrophages, and regulate inflammatory cytokine production by vascular smooth muscle cells (Figure 1).

In vitro studies have shown that PPARs have different functions depending on the cell type. For example, PPARa and PPARg are expressed in T-cells and control the production of a number of cytokines (i.e., interleukin-2, interferon) by inhibiting transcription pathways activating the inflammatory response. PPARs are also present in dendritic cells and appear to inhibit production of cytokines, such as interleukin 12, and thereby decreasing the type 1 TH-response. Finally, PPARs activation may also stabilize atherosclerotic plaques and inhibit the development of a thrombogenic response at the site of the plaque.

PPARs and the Inflammation Response: In Vivo Evidence
In animal studies, aortas of PPARa knockout mice display an exacerbated response to inflammation indicating that PPARa has anti-inflammatory properties. While clinical studies are limited, a study by Staels et al (Nature 1998; 393:790–793) comparing coronary arterial disease (CAD) patients with non-CAD patients have shown that CAD patients have significantly higher plasma IL-6 levels and these levels can be reduced by treatment with the PPARa activator, fenofibrate. The molecular mechanism of action for this effect is known as a process called transcriptional transrepression. In contrast, “the metabolic effects of PPARs occur primarily through a different mechanism which is called transactivation,” stated Dr. Staels.

In vivo evidence also suggests that PPARs may decrease the levels of inflammatory-dependent, acute phase proteins (fibrinogen and C-reactive protein) which are known risk factors for coronary heart disease and atherosclerosis.

PPARs and the Inflammation Response: Pathophysiological Relevance
In a recently published study, the effects of PPARg, g co-agonist was analyzed in a mouse model of atherosclerosis (Apo E deficient mice) and found this treatment to significantly lower the number of lesions. This study, as well as others, have shown that treatment with PPAR agonists can inhibit the development of atherosclerosis in addition to attenuating the development of diabetes.

Recent animal studies have also shown that a PPARa agonist can act as a neuroprotective agent in a model of stroke in mice. Since many diabetic patients have a higher risk of developing stroke, this may have an added beneficial effect during treatment for diabetes with PPAR agonists, although Dr. Staels warned that further studies in man are needed.

Dr. Staels ended his presentation by stating that the studies he discussed involved a large team of outstanding researchers and he thanked them for their dedication and hard work.

The Role of PPAR Gamma in Atherosclerosis and Restenosis

The high mortality rate of type 2 diabetic patients due to coronary heart disease (CHD) begins “in a prediabetic state where a cluster of metabolic abnormalities, insulin resistance, concomitant hyperinsulinemia, hypertension, and dyslipidemia lay the groundwork for the vascular damage that manifests itself many years later,” acknowledged Dr. Ronald Law, assistant professor of medicine at the University of California, Los Angeles, UCLA School of Medicine, Los Angeles, California. Furthermore, recent studies have shown that insulin resistance may be an important independent risk factor for the twofold increased risk of developing atherosclerosis in type 2 diabetic patients. These patients also have a twofold increased risk for developing restenosis compared to nondiabetic patients.

Atherosclerosis
In adipose tissue and skeletal muscle, the insulin signaling pathway initiates a cascade of biochemical events that converge on a glucose transporter, Glut-4. During insulin resistance, this pathway is attenuated. As a result, most of the Glut-4 in skeletal muscle and adipose tissue is sequestered in the cytoplasm. In contrast, when the insulin receptor is activated, the Glut-4 transporter migrates to plasma membranes where it extracts glucose from the circulation to skeletal muscle and adipose tissue for metabolism. The thiazolidinediones (TZDs) overcome this signaling defect in skeletal muscle and adipose tissue during insulin resistance and “they facilitate better recruitment of Glut-4 to the plasma membrane in response to insulin,” said Dr. Law.

PPARg is expressed in very high levels in adipose tissue and to a lesser extent in muscle tissue. More specifically, PPARg is present in vascular tissue, being expressed in endothelial cells, monocytes and macrophages and smooth muscle cells. The function of PPARg is unclear but animal models of atherosclerosis have begun to examine its role and found it may act as an anti-inflammatory agent. In one animal model, LDL receptor knockout mice given a high- fat diet developed lesions in the aortic arch within 3 months. Treatment with a PPARg ligand (troglitazone) reduced the number of lesions. Similar results were found in nondiabetic mice given a high- fructose diet (Arterioscler Thromb Vasc Biol 2001;21:365–371). These and other studies have suggested that “there may be a direct effect to suppress atherosclerosis by activating PPARg in the vessel wall independent of the metabolic abnormalities,” stated Dr. Law. In more aggressive models of atherosclerosis (infusion of angiotensin II), two other PPARg ligands (pioglitazone and rosiglitazone) significantly reduced (up to 60%) the number of lesions on vessel walls. The mechanism of action by which PPARg ligands inhibit atherosclerosis is still being investigated but a recent study indicates that PPARg inhibits the migration of monocytes by inhibiting the production of matrix metalloproteinase (Eur J Pharmacol 2000;401:259–270) (Figure 2). A second mechanism of action may also be involved since PPARg appears to block the early growth response gene 1 (EGR 1) which regulates many proinflammatory genes.

Restenosis
The animal models for restenosis are fairly straightforward. Investigators injure the vessel wall in normal rats and within 14 days a neointima develops. “If these animals were given troglitazone before injury and during the 14 days after injury, there was a very marked suppression of neointima formation: about a 60 percent reduction,” said Dr. Law. Similar results have been found with pioglitazone. “Now, what’s the mechanism?” asked Dr. Law. It appears that PPARg ligands inhibit migration of smooth muscle cells as well as the G1 g S progression of vascular smooth muscle cell cycle by inhibiting phosphorylation of retinoblastoma protein (Circulation 2000:101:1311–1318; J Biol Chem 2000;275:22435–22441).

Whether or not these animal studies will translate well into clinical trials remains to be seen but one study from Japan has shown that treatment with PPAR ligands results in a 40–50% reduction of intimal hyperplasia or restenosis in type 2 diabetic patients (J Am Coll Cardiol 2000;36:1529–1535).

Conclusion
Dr. Law ended his presentation by stating “TZD PPARg ligands may protect the vasculature from developing both atherosclerotic and restenotic lesions not only by normalizing metabolic abnormalities, insulin resistance, dyslipidemia, etc., but perhaps by directly activating PPARg express in the vessel wall which will ultimately inhibit both cell growth and movement.”