CL-316,243 Research Articles

Perilipin Targets a Novel Pool of Lipid Droplets for Lipolytic Attack by Hormone-sensitive Lipase

Adipocytes serve as the principal energy reservoir of the body; however, the subcellular organization of the machinery regulating lipid trafficking and metabolism is poorly understood. Mobilization of stored triglyceride is thought be controlled by interactions among intracellular lipases and proteins that coat lipid storage droplets. A major limitation of previous studies of hormone-mediated lipolysis, however, is the use of cultured model adipocytes whose three-dimensional architectures do not resemble those in real adipose tissue. To address this limitation, we investigated the intracellular targeting of perilipin, a major lipid coat protein, and hormone-sensitive lipase in three preparations that exhibit more appropriate morphologies: 3T3-L1 adipocytes grown in three-dimensional matrix, dissociated mature adipocytes from mouse adipose tissue, and adipocytes within intact fat pads. High resolution imaging of native and fluorescently tagged proteins indicate that: 1) perilipin preferentially targets a special class of peripheral lipid storage droplets, but not the major or central lipid storage droplets, 2) the peripheral droplets are the sites of attack by hormone-sensitive lipase, and 3) perilipin and hormone-sensitive lipase are continuously colocalized following lipolytic activation. These results indicate that in white adipose tissue, lipolysis takes place in a specialized subcellular domain that is distinct from the major lipid storage site and is defined by perilipin.

Metabolic and cellular plasticity in white adipose tissue II: role of peroxisome proliferator-activated receptor-α

Chronic activation of adipocyte beta-adrenergic receptors induces remodeling of white adipose tissue (WAT) that includes a transient inflammatory response followed by mitochondrial biogenesis, induction of fatty acid oxidation genes, and elevation of tissue oxidative metabolism. Gene profiling experiments of WAT during remodeling induced by the beta(3)-adrenergic receptor agonist CL-316,243 (CL) suggested that peroxisome proliferator-activated receptor-alpha (Ppara), which is upregulated by CL, might be an important transcriptional regulator of that process. Histological, physiological, and molecular analysis of CL-induced remodeling in wild-type mice and mice lacking Ppara demonstrated that Ppara was important for inducing adipocyte mitochondrial biogenesis and upregulating genes involved in fatty acid oxidation. Furthermore, Ppara-deficient mice exhibited sustained WAT inflammation during CL treatment, indicating that upregulation of Ppara limits proinflammatory signaling during chronic lipolytic activation. Together, these data support the hypothesis that WAT remodeling is an adaptive response to excessive fatty acid mobilization whereby Ppara and its downstream targets elevate fatty acid catabolism and suppress proinflammatory signaling.

Metabolic and cellular plasticity in white adipose tissue I: effects of β3-adrenergic receptor activation

Selective agonists of beta(3)-adrenergic receptors (Adrb3) exhibit potent anti-diabetes properties in rodent models when given chronically, yet the mechanisms involved are poorly understood. A salient feature of chronic Adrb3 activation is pronounced remodeling of white adipose tissue (WAT), which includes mitochondrial biogenesis and elevation of metabolic rate. To gain insights into potential mechanisms underlying WAT remodeling, the time course of remodeling induced by the Adrb3 agonist CL-316,243 (CL) was analyzed using histological, physiological, and global gene profiling approaches. The results indicate that continuous CL treatment induced a transient proinflammatory response that was followed by cellular proliferation among stromal cells and multilocular adipocytes. CL treatment strongly fragmented the central lipid storage droplet of mature adipocytes and induced mitochondrial biogenesis within these cells. Mitochondrial biogenesis was correlated with the upregulation of genes involved in fatty acid oxidation and mitochondrial electron transport activity. The elevated catabolic activity of WAT was temporally correlated with upregulation of peroxisome proliferator-activated receptor-alpha and its target genes, suggesting involvement of this transcription factor in coordinating the gene program that elevates WAT catabolic activity.

Multilocular fat cells in WAT of CL-316243-treated rats derive directly from white adipocytes.

Multilocular, mitochondria-rich adipocytes appear in white adipose tissue (WAT) of rats treated with the beta3-adrenoceptor agonist, CL-316243 (CL). Objectives were to determine whether these multilocular adipocytes derived from cells that already existed in the WAT or from proliferation of precursor cells and whether new mitochondria contained in them were typical brown adipocyte mitochondria. Use of 5-bromodeoxyuridine to identify cells that had undergone mitosis during the CL treatment showed that most multilocular cells derived from cells already present in the WAT. Morphological techniques showed that at least a subpopulation of unilocular adipocytes underwent conversion to multilocular mitochondria-rich adipocytes. A small proportion of multilocular adipocytes ( approximately 8%) was positive for UCP1 by immunohistochemistry. Biochemical techniques showed that mitochondrial protein recovered from WAT increased 10-fold and protein isolated from brown adipose tissue (BAT) doubled in CL-treated rats. Stained gels showed a different protein composition of new mitochondria isolated from WAT from that of mitochondria isolated from BAT. Western blotting showed new mitochondria in WAT to contain both UCP1, but at a much lower concentration than in BAT mitochondria, and UCP3, at a higher concentration than that in BAT mitochondria. We hypothesize that multilocular adipocytes present at 7 days of CL treatment have two origins. First, most come from convertible unilocular adipocytes that become multilocular and make many mitochondria that contain UCP3. Second, some come from a cell that gives rise to more typical brown adipocytes that express UCP1.

Mechanisms of the antidiabetic effects of the beta 3-adrenergic agonist CL-316243 in obese Zucker-ZDF rats.

Previous studies have demonstrated that chronic cold exposure activates the sympathetic nervous system, increases energy expenditure, improves glucose uptake in peripheral tissues [brown and white adipose tissues (BAT and WAT) and muscles] of normal rats. The goal of the present studies was to test whether the selective beta 3-adrenergic agonist CL-316243 (CL) would mimic the beneficial beneficial effects of cold exposure in lean and obese ZDF/Gmi-fa male (ZDF) rats, a new model of type II diabetes. In obese ZDF rats, chronic infusion of CL (1 mg.kg-1.day-1 for 14 days) significantly decreased body weight gain, food intake, and WAT weight. It also increased total tissue cytochrome oxidase activity, not only in BAT (15 times), but also in WAT (2-4) times, suggesting that it progressively enhanced mitochondriogenesis in adipose tissues. CL treatment normalized hyperglycemia and reduced hyperinsulinemia and circulating free fatty acid (FFA) levels. It also improved glucose tolerance and reduced insulin response during an intravenous glucose tolerance test. In general, the beneficial effects of CL were more pronounced in obese than in lean rats. Hyperinsulinemic-euglycemic glucose clamps combined with the [2-3H]deoxyglucose method revealed that CL markedly improved insulin responsiveness in obese rats (3-4 times) and increased glucose uptake in BAT (21 times), WAT (3 times), skeletal muscles (2-3 times), and in the diaphragm (2.8 times), but not in the heart. It is concluded that chronic CL treatment improves glucose tolerance and insulin responsiveness in obese ZDF rats by a mechanism similar to that induced by chronic cold exposure, i.e., by stimulating facultative thermaogenesis, mitochondriogenesis, and glucose utilization in BAT and WAT. In addition to this mechanism, the reduction in plasma FFA levels induced by chronic CL treatment may further contribute to enhance glucose uptake in skeletal muscles (a tissue that does not express typical beta 3-adrenoceptors) via the "glucose-fatty acid" cycle. The antiobesity and antidiabetic properties of CL suggest that selective beta 3-adrenergic agonists may represent useful agents for the treatment of type II diabetes.

Beta 3-Adrenergic agonist induces a functionally active uncoupling protein in fat and slow-twitch muscle fibers.

The mitochondrial uncoupling protein (UCP) has usually been found only in brown adipose tissue. We recently observed that a chronic administration of the beta 3-adrenergic agonist CL-316,243 (CL) induced the ectopic expression of UCP in white fat and skeletal muscle in genetic obese yellow KK mice. The aim of the present study was to examine whether UCP could be induced in nongenetic obese animals produced by neonatal injections of monosodium L-glutamate (MSG). The daily subcutaneous injection of CL (0.1 mg/kg) to MSG-induced obese mice for 2 wk caused significant reductions of body weight (15%) and white fat pad weight (58%). Northern and Western blot analyses showed that CL induced significant expressions of UCP in the white fat and muscle, as well as in brown fat. Immunohistochemical observations revealed that the UCP stains in white fat were localized on multilocular cells and that those in muscle were localized on slow-twitch fibers rich in mitochondria. Immunoelectron microscopy confirmed the mitochondrial localization of UCP in the myocytes. The guanosine 5'-diphosphate (GDP) binding to mitochondria in brown fat doubled after the CL treatment. Moreover, significant GDP binding was detected in the white fat and muscle of the CL-treated mice, at about one-fourth and one-thirteenth the activity of brown fat, respectively, suggesting that ectopically expressed UCP is functionally active. We concluded that the beta 3-adrenergic agonist CL can induce functionally active UCP in white fat and slow-twitch muscle fibers of obese mice.

Suppression of insulin-stimulated phosphatidylinositol 3-kinase activity by the β 3-adrenoceptor agonist CL316243 in rat adipocytes

Insulin increased 2-deoxyglucose (2-DG) uptake via the translocation of glucose transporter (GLUT) 4 to the plasma membrane fraction in rat adipocytes. The stimulatory actions of insulin were accompanied by both an increase in the immunoreactive p85 subunit of phosphatidylinositol (PI) 3-kinase in the plasma membrane fractions and PI 3-kinase activation by tyrosine phosphorylation of the p85 subunit. The β 3-adrenoceptor agonist CL316243 (CL) suppressed all the insulin actions in adenosine deaminase (ADA)-treated cells, but was without effect in non-ADA-treated cells. The inhibitory effects of CL on GLUT 4 translocation and PI 3-kinase activation were abolished by the addition of N 6-phenylisopropyl adenosine. Cholera toxin treatment, which markedly increased intracellular cAMP levels, suppressed increases in the levels of GLUT 4 and PI 3-kinase in the plasma membrane fractions in response to insulin. In addition, dibutyryl (Bt 2) cAMP also impaired the activation of PI 3-kinase by insulin. These results indicated that CL suppressed insulin-stimulated glucose transport under conditions where cAMP levels were markedly increased (∼12-fold). The inhibitory actions of PI 3-kinase activation by insulin were exerted even when cAMP, 8-bromo-cAMP, or Bt 2 cAMP was added to immunoprecipitates of the p85 subunit of PI 3-kinase, after treating the cells with insulin. These results suggest that CL suppressed insulin-stimulated PI 3-kinase activity via a cAMP-dependent mechanism, at least in part, direct cAMP action in ADA-treated adipocytes, by which PI 3-kinase activation was inhibited, resulting in the decrease in GLUT 4 translocation and subsequent 2-DG uptake in response to insulin.

Effect of CL-316,243, a thermogenic beta 3-agonist, on energy balance and brown and white adipose tissues in rats.

The objective was to assess the effect of a new, highly selective beta 3-adrenergic agonist, CL-316,243 (CL) (J. D. Bloom, M. D. Dutia, B. D. Johnson, A. Wissner, M. G. Burns, E. E. Largis, J. A. Dolan, and T. H. Claus., J. Med. Chem. 35: 3081, 1992), on energy balance and brown and white adipose tissues (BAT and WAT, respectively) in young rats eating a high-fat diet to induce obesity. Chronic treatment with CL increased body temperature and 24-h energy expenditure, mainly by increasing resting metabolic rate. Food intake was not altered but carcass fat was reduced. Interscapular BAT was markedly hypertrophied, with three- to fourfold increases in the content of uncoupling protein (UCP) and cytochrome oxidase. Quantitative immunoelectron microscopy of interscapular BAT of CL-treated rats showed smaller mitochondria with an unchanged total amount of UCP per mitochondrion. The relative frequency of the four major cell types in BAT (mature brown adipocytes, preadipocytes, interstitial cells, endothelial cells) was not altered. The CL-induced hypertrophy differed from that induced by chronic stimulation by endogenous norepinephrine (as in cold-adaptation) in absence of hyperplasia (there was a slightly reduced DNA content), absence of an increase in the thyroxine (T4) 5'-deiodinase activity, and absence of a selective increase in UCP concentration. WAT depots weighed less and had fewer cells (lower DNA content) in the CL-treated rats. Some multilocular adipocytes appeared in these normally almost exclusively unilocular WAT depots (mesenteric, inguinal, epididymal, retroperitoneal). We conclude that CL not only promotes BAT mitochondrial proliferation and thermogenesis and overall energy expenditure and leanness, but also retards the development of WAT hyperplasia during the early stage of diet-induced obesity.