Novel Receptors and Their Functions Define Pathways of Lipid Metabolism and Mechanisms of Ather
Novel Receptors and Their Functions Define Pathways of Lipid Metabolism and Mechanisms of Atherosclerosis
Introduction
The metabolic syndrome is by now unequivocally established in the minds of endocrinologists as a common and burgeoning disease linked to premature atherosclerosis and cardiovascular disease. Many symposia and presentations at ENDO 2003 described the clinical features and changing epidemiology of this epidemic illness. However, although the metabolic syndrome now has a more uniform definition, its causes remain unclear. There are numerous molecules that appear to be reasonable culprits as demonstrated in animal models and cellular studies, for example, adipokines and cytokines such as adiponectin and tumor necrosis factor alpha and enzymes such as 11-beta hydroxysteroid dehydrogenase type 1. However, their relevance in human disease and, perhaps more important, their relative contribution to the overall disease state remain open questions.
In contrast, the function of nuclear receptors of the peroxisome proliferator activator (PPAR) family is clearly both highly relevant to human lipid physiology and disease and of proven significance through the disease-modifying efficacy of its therapeutically used ligands. In a plenary session entitled "Atherosclerosis: Lipids and Nuclear Receptors," Ronald M. Evans, PhD, of the Salk Institute, La Jolla, California, gave an excellent overview of the central role of the PPAR family in lipid metabolism and focused on new insights into the important functions of the hitherto poorly understood PPAR-delta receptor.
PPAR-Gamma
As originally described in the laboratory of Dr. B. Spiegelman at the Dana Farber Cancer Institute, Boston, Massachusetts, PPAR-gamma is critical in adipocyte development. This transcription factor is expressed early in the differentiation of preadipocytes. However, numerous subsequent studies have shown that it is also important in the proper maintenance of function in the mature adipocyte, for example, in the ongoing expression of genes encoding lipid oxidative enzymes.
More recent functions delineated in the Evans laboratory include its role in the differentiation of monocytic/myeloid cells. This dual effect on both adipocytes and monocytes places PPAR-gamma squarely in the regulatory center of cardiovascular atherosclerosis. Lipid-laden macrophages in vessel walls are a pathognomonic feature of atherosclerosis. These macrophages express PPAR-gamma, which in turn induces the expression of the cholesterol scavenger receptor CD36. These cells are also associated with vessel wall inflammation and increased entry of monocytic cells containing a high proportion of oxidized low-density lipoprotein (LDL). Oxidized LDL particles harbor PPAR-gamma ligands. These findings reveal a physiologic, protective cycle, termed the gamma cycle, that can defuse the potential toxicity of lipid-laden macrophages in the vessel wall. Thus, oxidized LDL provides ligands that can activate PPAR-gamma, which in turn increases the expression of factors required for fatty acid uptake and storage such as lipoprotein lipase and CD36. At the same time, PPAR-gamma activation also induces activation of the LXR gene, which increases expression of the gene for ABCA-1, the chief regulator of the reverse cholesterol process. This then removes cholesterol moieties from cells and exports them in the "safe" form of high-density lipoprotein cholesterol.
Thus, the central pathway of macrophage/LDL-mediated vessel wall damage carries within it the potential for recovery. This potential can be exploited by the use of PPAR-gamma agonists, which have been shown to reverse atherosclerotic lesions in animals. The significance of this effect is further underscored by the fact that macrophages lacking PPAR-gamma cause acceleration of atherosclerotic plaques.
PPAR-Delta
PPAR-gamma is an important key to LDL-cholesterol regulation. But what of that other cardiovascular risk-elevating culprit in the metabolic syndrome, the very low-density lipoprotein (VLDL) lipid--containing particle? Dr. Evans described exciting data from his laboratory that detail the role of PPAR-delta in regulating this other potentiator of dyslipidemia and atherosclerosis.
In brief, PPAR-delta is a "sensor" of VLDL, with a central physiologic function in triglyceride metabolism and protection from VLDL-mediated inflammation in an atherosclerotic lesion. VLDL is a triglyceride-rich particle that harbors specific ligands for PPAR-delta. To dissect its function, the Evans group engineered embryonic stem cells lacking PPAR-delta and placed them in mice that were irradiated and given a bone marrow transplant. On a high-fat diet, the PPAR-delta null mice had a slower progression of atherosclerotic lesions than the wild-type mice. Specifically, inflammatory markers in the lesions were reduced, as illustrated by a diminution in the expression of the chemotactic protein MCP-1. When PPAR-delta was reintroduced into the null mice, a remarkable effect was observed: replacement of the transcription factor increased expression of the inflammatory marker in the absence of ligand but decreased it in the presence of ligand. The proposed mechanism for this effect is that in the nonliganded state PPAR-delta sequesters BCL-6, a repressor of MCP-1 transcription, thus permitting unfettered expression of MCP-1. In the liganded state, PPAR-delta cannot sequester BCL-6; hence, MCP-1 expression is muted.
To examine the full spectrum of PPAR-delta effects, PPAR-delta transgenic mice were constructed, and these have a lean phenotype. Conversely, PPAR-delta knockout mice are prone to diet-induced obesity. The PPAR-delta overexpressing mice have decreased fat stores due to increased fatty acid oxidation and uncoupling of oxidative phosphorylation in both white adipose tissues and brown adipose tissue. These mechanisms result in significant reduction in circulating triglycerides and free fatty acids but not in plasma cholesterol level.
These results reveal a delicate physiologic dance of cholesterol transfer in macrophages that involves the coordinated influx and efflux of cholesterol together with the attenuation of inflammation. PPAR-gamma and PPAR-delta regulate this dance to maintain homeostasis in the vessel wall in the face of dietary and genetic variations. At the level of overall fat balance, PPAR-gamma acts as a prime mover of appropriate and effective lipid storage in adipocytes, whereas PPAR-delta polices the pathway of fatty acid disposal by oxidation.
The balance among lipid accumulation, storage, and utilization is so critical that the body appears to have multiple intercrossing and overlapping pathways to regulate it. The chief organs that subserve these pathways are the small intestine, liver, and adipose depots. To protect against marked surges or decreases of serum cholesterol levels in the face of marked variations in dietary sterols, interactions between the small intestine and the liver are particularly important.
A Novel Pair of Receptors: ABCG-5 and ABCG-8
Helen H. Hobbs, MD, of the University of Texas Southwestern Medical School, Dallas, presented an informative lecture at the same plenary session in which she discussed the function of a novel pair of receptors in the gut and liver, ABCG-5 and ABCG-8, in protecting the body against sterol accumulation. The functions of these receptors were revealed through her investigations of the differences in the splanchnic handling of animal-derived sterols (eg, cholesterol) vs plant-derived sterols (eg, sitosterol) and of an unusual illness, sitosterolemia.
Circulating levels of total and LDL-cholesterol vary throughout a 2-fold range in humans. Genetically derived factors appear to account for approximately 50% of these levels in a given individual, whereas dietary factors appear to account for the remaining half. The dietary factors include not only the total calories consumed but also the amounts and proportions of the various types of fats and sterols in the diet. Normally, there is a large discrepancy between the levels of cholesterol and sitosterol in the blood, approximately 120-200 mg/dL of the former and < 1 mg/dL of the latter, despite the fact that up to one third of the sterols in the average US diet consist of sitosterol. The discrepancy results from marked differences in the fractional intestinal absorption and biliary excretion of the 2 sterols. However, in the condition termed sitosterolemia, sitosterol levels are 15-30 times higher than normal, in association with xanthomas and elevated cardiovascular risk. Uniquely, it is extremely responsive to treatment by a low-cholesterol diet or bile acid resins. Biochemically, the condition can be traced to 2 defects: a marked increase in small intestinal cholesterol absorption (together with an increased fractional absorption of sitosterol) and a markedly diminished ability to secrete sterols into the bile. This finding implied defects in the transfer of the sterols from the gut to the liver and then back again from the liver to the gut. But what normally regulates these transfer steps?
Several lines of evidence suggest that the nuclear receptor LXR, which heterodimerizes with RXR to regulate gene transcription, is involved in both steps. For example, treatment of mice with an LXR agonist decreases intestinal absorption of dietary cholesterol and increases sterol excretion in the bile. Dr. Hobbs' laboratory therefore searched for genes targeted by LXR to uncover genetic factors regulating cholesterol absorption and excretion. They found 2 half-transporter molecules of the "ATP-cassette" type, ABCG-5 and ABCG-8, which are coordinately expressed from 2 tandem genes exclusively in hepatocytes and enterocytes. Notably, patients with sitosterolemia harbored mutations in one or the other of these genes. Further studies revealed that the ABCG-5/8 receptors in the basal surface of the enterocyte function to pick up a portion of absorbed sterols (preferentially plant sterols) and send them back into the intestinal lumen. The same receptors, on the luminal surface of hepatocytes in the liver lobules, function to excrete sterols into the bile. These mechanisms could be demonstrated in vivo, since transgenic mice overexpressing ABCG-5/8 markedly increased biliary excretion of sterols. These mice also had slightly diminished circulating sterol concentrations despite a marked (presumably compensatory) increase in de novo hepatic sterol synthesis. Conversely, ABCG-5/8 "knockout" mice recapitulated the phenotype of sitosterolemia, with decreased biliary secretion and increased circulating levels of total cholesterol and plant sterols.
These studies provide important insights into the interactions between genetic and environmental mechanisms in the response to fluctuations in dietary fat and sterol loads. The ABCG-5/8 receptors modulate fluxes of dietary animal and plant sterols to keep plasma cholesterol levels steady. They also establish the points of splanchnic entry and exit of sterols as key therapeutic targets for hypercholesterolemic states, suggesting that the older bile acid resins and the new drug ezetimibe may be particularly useful in persons with structural or functional defects in these transfer mechanisms. Of course, the receptors themselves would be molecular targets for novel drug development to treat hypercholesterolemia.
References
The metabolic syndrome is by now unequivocally established in the minds of endocrinologists as a common and burgeoning disease linked to premature atherosclerosis and cardiovascular disease. Many symposia and presentations at ENDO 2003 described the clinical features and changing epidemiology of this epidemic illness. However, although the metabolic syndrome now has a more uniform definition, its causes remain unclear. There are numerous molecules that appear to be reasonable culprits as demonstrated in animal models and cellular studies, for example, adipokines and cytokines such as adiponectin and tumor necrosis factor alpha and enzymes such as 11-beta hydroxysteroid dehydrogenase type 1. However, their relevance in human disease and, perhaps more important, their relative contribution to the overall disease state remain open questions.
In contrast, the function of nuclear receptors of the peroxisome proliferator activator (PPAR) family is clearly both highly relevant to human lipid physiology and disease and of proven significance through the disease-modifying efficacy of its therapeutically used ligands. In a plenary session entitled "Atherosclerosis: Lipids and Nuclear Receptors," Ronald M. Evans, PhD, of the Salk Institute, La Jolla, California, gave an excellent overview of the central role of the PPAR family in lipid metabolism and focused on new insights into the important functions of the hitherto poorly understood PPAR-delta receptor.
PPAR-Gamma
As originally described in the laboratory of Dr. B. Spiegelman at the Dana Farber Cancer Institute, Boston, Massachusetts, PPAR-gamma is critical in adipocyte development. This transcription factor is expressed early in the differentiation of preadipocytes. However, numerous subsequent studies have shown that it is also important in the proper maintenance of function in the mature adipocyte, for example, in the ongoing expression of genes encoding lipid oxidative enzymes.
More recent functions delineated in the Evans laboratory include its role in the differentiation of monocytic/myeloid cells. This dual effect on both adipocytes and monocytes places PPAR-gamma squarely in the regulatory center of cardiovascular atherosclerosis. Lipid-laden macrophages in vessel walls are a pathognomonic feature of atherosclerosis. These macrophages express PPAR-gamma, which in turn induces the expression of the cholesterol scavenger receptor CD36. These cells are also associated with vessel wall inflammation and increased entry of monocytic cells containing a high proportion of oxidized low-density lipoprotein (LDL). Oxidized LDL particles harbor PPAR-gamma ligands. These findings reveal a physiologic, protective cycle, termed the gamma cycle, that can defuse the potential toxicity of lipid-laden macrophages in the vessel wall. Thus, oxidized LDL provides ligands that can activate PPAR-gamma, which in turn increases the expression of factors required for fatty acid uptake and storage such as lipoprotein lipase and CD36. At the same time, PPAR-gamma activation also induces activation of the LXR gene, which increases expression of the gene for ABCA-1, the chief regulator of the reverse cholesterol process. This then removes cholesterol moieties from cells and exports them in the "safe" form of high-density lipoprotein cholesterol.
Thus, the central pathway of macrophage/LDL-mediated vessel wall damage carries within it the potential for recovery. This potential can be exploited by the use of PPAR-gamma agonists, which have been shown to reverse atherosclerotic lesions in animals. The significance of this effect is further underscored by the fact that macrophages lacking PPAR-gamma cause acceleration of atherosclerotic plaques.
PPAR-Delta
PPAR-gamma is an important key to LDL-cholesterol regulation. But what of that other cardiovascular risk-elevating culprit in the metabolic syndrome, the very low-density lipoprotein (VLDL) lipid--containing particle? Dr. Evans described exciting data from his laboratory that detail the role of PPAR-delta in regulating this other potentiator of dyslipidemia and atherosclerosis.
In brief, PPAR-delta is a "sensor" of VLDL, with a central physiologic function in triglyceride metabolism and protection from VLDL-mediated inflammation in an atherosclerotic lesion. VLDL is a triglyceride-rich particle that harbors specific ligands for PPAR-delta. To dissect its function, the Evans group engineered embryonic stem cells lacking PPAR-delta and placed them in mice that were irradiated and given a bone marrow transplant. On a high-fat diet, the PPAR-delta null mice had a slower progression of atherosclerotic lesions than the wild-type mice. Specifically, inflammatory markers in the lesions were reduced, as illustrated by a diminution in the expression of the chemotactic protein MCP-1. When PPAR-delta was reintroduced into the null mice, a remarkable effect was observed: replacement of the transcription factor increased expression of the inflammatory marker in the absence of ligand but decreased it in the presence of ligand. The proposed mechanism for this effect is that in the nonliganded state PPAR-delta sequesters BCL-6, a repressor of MCP-1 transcription, thus permitting unfettered expression of MCP-1. In the liganded state, PPAR-delta cannot sequester BCL-6; hence, MCP-1 expression is muted.
To examine the full spectrum of PPAR-delta effects, PPAR-delta transgenic mice were constructed, and these have a lean phenotype. Conversely, PPAR-delta knockout mice are prone to diet-induced obesity. The PPAR-delta overexpressing mice have decreased fat stores due to increased fatty acid oxidation and uncoupling of oxidative phosphorylation in both white adipose tissues and brown adipose tissue. These mechanisms result in significant reduction in circulating triglycerides and free fatty acids but not in plasma cholesterol level.
These results reveal a delicate physiologic dance of cholesterol transfer in macrophages that involves the coordinated influx and efflux of cholesterol together with the attenuation of inflammation. PPAR-gamma and PPAR-delta regulate this dance to maintain homeostasis in the vessel wall in the face of dietary and genetic variations. At the level of overall fat balance, PPAR-gamma acts as a prime mover of appropriate and effective lipid storage in adipocytes, whereas PPAR-delta polices the pathway of fatty acid disposal by oxidation.
The balance among lipid accumulation, storage, and utilization is so critical that the body appears to have multiple intercrossing and overlapping pathways to regulate it. The chief organs that subserve these pathways are the small intestine, liver, and adipose depots. To protect against marked surges or decreases of serum cholesterol levels in the face of marked variations in dietary sterols, interactions between the small intestine and the liver are particularly important.
A Novel Pair of Receptors: ABCG-5 and ABCG-8
Helen H. Hobbs, MD, of the University of Texas Southwestern Medical School, Dallas, presented an informative lecture at the same plenary session in which she discussed the function of a novel pair of receptors in the gut and liver, ABCG-5 and ABCG-8, in protecting the body against sterol accumulation. The functions of these receptors were revealed through her investigations of the differences in the splanchnic handling of animal-derived sterols (eg, cholesterol) vs plant-derived sterols (eg, sitosterol) and of an unusual illness, sitosterolemia.
Circulating levels of total and LDL-cholesterol vary throughout a 2-fold range in humans. Genetically derived factors appear to account for approximately 50% of these levels in a given individual, whereas dietary factors appear to account for the remaining half. The dietary factors include not only the total calories consumed but also the amounts and proportions of the various types of fats and sterols in the diet. Normally, there is a large discrepancy between the levels of cholesterol and sitosterol in the blood, approximately 120-200 mg/dL of the former and < 1 mg/dL of the latter, despite the fact that up to one third of the sterols in the average US diet consist of sitosterol. The discrepancy results from marked differences in the fractional intestinal absorption and biliary excretion of the 2 sterols. However, in the condition termed sitosterolemia, sitosterol levels are 15-30 times higher than normal, in association with xanthomas and elevated cardiovascular risk. Uniquely, it is extremely responsive to treatment by a low-cholesterol diet or bile acid resins. Biochemically, the condition can be traced to 2 defects: a marked increase in small intestinal cholesterol absorption (together with an increased fractional absorption of sitosterol) and a markedly diminished ability to secrete sterols into the bile. This finding implied defects in the transfer of the sterols from the gut to the liver and then back again from the liver to the gut. But what normally regulates these transfer steps?
Several lines of evidence suggest that the nuclear receptor LXR, which heterodimerizes with RXR to regulate gene transcription, is involved in both steps. For example, treatment of mice with an LXR agonist decreases intestinal absorption of dietary cholesterol and increases sterol excretion in the bile. Dr. Hobbs' laboratory therefore searched for genes targeted by LXR to uncover genetic factors regulating cholesterol absorption and excretion. They found 2 half-transporter molecules of the "ATP-cassette" type, ABCG-5 and ABCG-8, which are coordinately expressed from 2 tandem genes exclusively in hepatocytes and enterocytes. Notably, patients with sitosterolemia harbored mutations in one or the other of these genes. Further studies revealed that the ABCG-5/8 receptors in the basal surface of the enterocyte function to pick up a portion of absorbed sterols (preferentially plant sterols) and send them back into the intestinal lumen. The same receptors, on the luminal surface of hepatocytes in the liver lobules, function to excrete sterols into the bile. These mechanisms could be demonstrated in vivo, since transgenic mice overexpressing ABCG-5/8 markedly increased biliary excretion of sterols. These mice also had slightly diminished circulating sterol concentrations despite a marked (presumably compensatory) increase in de novo hepatic sterol synthesis. Conversely, ABCG-5/8 "knockout" mice recapitulated the phenotype of sitosterolemia, with decreased biliary secretion and increased circulating levels of total cholesterol and plant sterols.
These studies provide important insights into the interactions between genetic and environmental mechanisms in the response to fluctuations in dietary fat and sterol loads. The ABCG-5/8 receptors modulate fluxes of dietary animal and plant sterols to keep plasma cholesterol levels steady. They also establish the points of splanchnic entry and exit of sterols as key therapeutic targets for hypercholesterolemic states, suggesting that the older bile acid resins and the new drug ezetimibe may be particularly useful in persons with structural or functional defects in these transfer mechanisms. Of course, the receptors themselves would be molecular targets for novel drug development to treat hypercholesterolemia.
References
Evans R, Lee C-H, Wang Y, et al. PPARs and the complex journey to obesity. Program and abstracts of the 85th annual meeting of the Endocrine Society; June 19-22, 2003; Philadelphia, Pennsylvania.
Hobbs H. Genetic protection against cholesterol accumulation and atherosclerosis. Program and abstracts of the 85th annual meeting of the Endocrine Society; June 19-22, 2003; Philadelphia, Pennsylvania.