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Increased rates of fatty acid uptake and plasmalemmal fatty acid transporters in obese Zucker rats.

Giant vesicles were used to study the rates of uptake of long-chain fatty acids by heart, skeletal muscle, and adipose tissue of obese and lean Zucker rats. With obesity there was an increase in vesicular fatty acid uptake of 1.8-fold in heart, muscle and adipose tissue. In some tissues only fatty acid translocase (FAT) mRNA (heart, +37%; adipose, +80%) and fatty acid-binding protein (FABPpm) mRNA (heart, +148%; adipose, +196%) were increased. At the protein level FABPpm expression was not changed in any tissues except muscle (+14%), and FAT/CD36 protein content was altered slightly in adipose tissue (+26%). In marked contrast, the plasma membrane FAT/CD36 protein was increased in heart (+60%), muscle (+80%), and adipose tissue (+50%). The plasma membrane FABPpm was altered only in heart (+50%) and adipose tissues (+70%). Thus, in obesity, alterations in fatty acid transport in metabolically important tissues are not associated with changes in fatty acid transporter mRNAs or altered fatty acid transport protein expression but with their increased abundance at the plasma membrane. We speculate that in obesity fatty acid transporters are relocated from an intracellular pool to the plasma membrane in heart, muscle, and adipose tissues.

Fatty acids (FA) 1 are important substrates for most mammalian tissues. Based on their hydrophobic structure it has been postulated that FA are sequestered by cells through passive diffusion across the plasma membrane (cf. Ref. 1). However, other evidence has shown that FA also traverse the plasma membrane via a protein-mediated mechanism (cf. Refs. 2 and 3). Indeed, this latter system is quantitatively more important than passive diffusion, as FA uptake can be reduced markedly by inhibitors of protein-mediated membrane transport (4 -6) and by a reactive ester of oleate (4). Thus, a number of groups began to search for FA transport proteins.
Several putative fatty acid transport proteins have been identified that promote the cellular uptake of FA. These are a 43-kDa plasma membrane fatty acid-binding protein (FABPpm) (7), identical to mitochondrial aspartate aminotransferase (7)(8)(9), and an 88-kDa heavily glycosylated fatty acid translocase (FAT/CD36), the rat homologue of human CD36 (10). In addition, to these membrane-associated proteins, a soluble cytoplasmic fatty acid-binding protein (FABPc) is also important for cellular FA uptake, because in FABPc null mice there is a marked decrease of FA influx into cardiac myocytes (11). FATP1, another putative fatty acid transport protein (12,13), correlates inversely with fatty acid transport in muscle and heart (4), and this protein appears to be a very long-chain acyl-CoA synthetase (14,15). These observations suggest that FATP1 is unlikely to be involved directly in fatty acid translocation across the plasma membrane.
To examine the regulation of transmembrane fatty acid transport, we have characterized giant vesicles (4,16,17), which can be prepared from metabolically important tissue such as heart (4) and skeletal muscle (16,17) as well as adipose tissue (present study). These giant vesicles are oriented rightside-out, and they contain the cytosolic fatty acid-binding protein, FABPc, which acts as a FA sink (4,16) Importantly, in giant vesicles, FA uptake across the plasma membrane can be examined in the absence of any confounding effects of FA metabolism (4,16,17), which has been a problem in studies using respiring cells such as adipocytes (18,19), hepatocytes (20,21), and cardiac myocytes (6,22). With giant vesicles we have shown that fatty acid transport can be increased (a) when the number of FAT/CD36 transport proteins are increased over the course of several days (23) or (b) when the FAT/CD36 protein is translocated, within minutes, from an intracellular pool to the plasma membrane (17).
It is becoming increasingly evident that disorders in lipid metabolism are an important factor contributing to the etiology of a number of common human diseases such as diabetes and obesity (cf. Ref. 24). Our evidence that protein-mediated FA uptake is a rate-limiting step in lipid metabolism allows the attractive speculation that malfunctioning of the FA uptake process could be a common critical factor in the pathogenesis of these diseases. The obese Zucker rat, a widely used rodent model for obesity and insulin resistance, has a mutation in the gene encoding the receptor for leptin. Homozygosity for this mutation is responsible for alterations in body lipid homeostasis resulting in the accumulation of body fat mass (25). Recent studies using obese Zucker rats (19) and other rodent models of obesity (26,27) have shown tissue-specific changes in FA transporters at the mRNA and FA utilization levels (i.e. fatty acid uptake ϩ metabolism). Up-regulation of FA utilization and transporter mRNAs in adipocytes of obese Zucker rats occur early in life preceding peripheral insulin resistance and obesity, suggesting that accumulation of FA transporters in the plasma membrane of adipocytes may be a critical step leading to obesity (19).
However, these changes in the accumulation of transporters measured at the mRNA level only (19,26,27) do not provide evidence that similar changes are occurring either at the protein level or in FA uptake across the plasma membrane (vesicular FA transport). Measurements of FA uptake in isolated cell systems are confounded by the subsequent rapid activation of FA to their CoA esters (19,26). Importantly, our discovery of an intracellular storage pool of FAT/CD36 (17) suggests that in addition to determining the total amount of FA transporters, their subcellular localization also needs to be considered so as to link properly the involvement of FA transporters with FA uptake rates. Therefore, to examine tissue-specific changes in vesicular fatty acid transport and the subcellular location of FA transporters in obese Zucker rats: (a) we examined the levels of the two functionally important FA transporters (FAT/CD36 and FABPpm), both at the mRNA and protein level, as well as FABPc in metabolically important tissues (heart, muscle, adipose tissue); (b) we measured FA uptake rates in these tissues using giant vesicles in which FA uptake can be measured in the complete absence of FA metabolism; and (c) because giant vesicles have been proven suitable representatives of the plasma membrane (17,28), we used these vesicles to provide information on the plasmalemmal localization of FA transporters. Our studies demonstrate that in obesity vesicular FA uptake rates are markedly increased, but this is generally not associated with concomitant changes in the expression of fatty acid transporters. Instead, the increases in fatty acid transport are associated with changes in plasmalemmal fatty acid transporters.
Animals-Female obese (fa/fa) and lean (fa/ϩ) Zucker rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). At 3 months of age, they were anesthetized with an intraperitoneal injection of Somnotol. Subsequently, hind limb muscle, epididymal adipose tissue, and heart were removed for FA uptake studies and for determination of FA transporters.
Metabolite Assays-Blood samples were collected into NaF/heparinized microcentrifuge tubes and centrifuged in a microcentrifuge. Plasma was then separated from red blood cells and stored at Ϫ80°C until use. Glucose was determined by a spectrophotometric method (Sigma-Aldrich). Insulin was determined by radioimmunoassay using a rat-specific antibody (Linco, St. Charles, MO). Plasma fatty acids and triacylglycerols were determined using spectrophotometric procedures (Wako Chemicals, Richmond, VA, and Sigma-Aldrich, respectively).
Isolation of Giant Vesicles-Giant vesicles from skeletal muscle and heart were isolated as described previously (4,17,29). Minor modifications were introduced to prepare giant vesicles from adipose tissue.
Briefly, all of the tissues were cut into thin layers (1-3 mm thick) and incubated for 1 h at 34°C in 140 mM KCl, 10 mM MOPS (pH 7.4), aprotinin (10 mg/ml), and collagenase in a shaking water bath. Collagenase type VII (150 units/ml) was used for muscle tissue, collagenase type II (0.3%, w/v) was used for heart, and collagenase type 2a (0.05%, w/v) was used for adipose tissue. At the end of the incubation, the supernatant was collected, and the remaining tissue was washed with KCl/MOPS and 10 mM EDTA, which resulted in a second supernatant. Both supernatant fractions were pooled, and Percoll and aprotinin were added to final concentrations of 16% (v/v) and 10 mg/ml, respectively. The resulting suspension was placed at the bottom of a density gradient consisting of a 3-ml middle layer of 4% Nycodenz (w/v) and a 1-ml KCl/MOPS upper layer. This sample was centrifuged at 60 ϫ g for 45 min at room temperature. Subsequently, the vesicles were harvested from the interface of the upper and middle layers, diluted in KCl/MOPS, and recentrifuged at 900 ϫ g for 10 min. In case of skeletal muscle, the pellet was resuspended in KCl/MOPS to a protein concentration of 2-3 mg/ml, in case of the other tissues, the pellet was resuspended to a protein concentration of 0.4 -0.8 mg/ml.
Palmitate Uptake by Giant Vesicles-Palmitate uptake studies were performed as we have described previously (4,16,23). Briefly, 40 l of 0.1% bovine serum albumin in KCl/MOPS containing unlabeled (15 M) and radiolabeled 0.3 Ci of [ 3 H]palmitate and 0.06 Ci of [ 14 C]mannitol were added to 40 l of vesicle suspension. The incubation was carried out for 15 s. Palmitate uptake was terminated by the addition of 1.4 ml of ice-cold KCl/MOPS, 2.5 mM HgCl 2 , and 0.1% bovine serum albumin. The sample was then quickly centrifuged in a microcentrifuge at 12,000 rpm for 1 min. The supernatant was discarded, and radioactivity was determined in the tip of the tube. Nonspecific uptake was measured by adding the stop solution prior to addition of the radiolabeled palmitate solution.
Northern and Western Blotting-mRNA abundance of FAT and FABPpm were determined as described previously (30). The cDNA For FAT/CD36 (10) was kindly provided by Dr. N. A. Abumrad (State University of New York, Stony Brook, NY), and the cDNA for mitochondrial aspartate aminotransferase/FABPpm (31) was a gift from Dr. A. Iriarte (University of Missouri). FAT/CD36 and FABPpm protein content were determined in both homogenates and giant vesicles prepared from heart and muscle as we have described previously (4,16,17,23,32) and from adipose tissue using essentially the same procedures. For detection of FAT/CD36 and FABPpm, we used a monoclonal antibody (MO25) against human CD36 (33) and a rabbit polyclonal anti-FABPpm antiserum, respectively, as described previously (4,17,34). For detection of monocarboxylate transporter 1 (MCT1), we used a polyclonal antibody (32).
Cytoplasmic H-FABP Determinations-The contents of heart-type cytoplasmic FABP (H-FABPc) in homogenates and giant vesicles from heart and skeletal muscles were determined by a sandwich-type enzyme-linked immunosorbent assay as previously described (35,36). Because of the unavailability of the appropriate antibody we were not able to determine the adipocyte lipid binding protein (A-LBP), the predominant FABPc in adipose tissue.
Vesicle Sizing-Giant vesicles were prepared from heart, muscle and adipose tissue from lean rats and obese Zucker rats. These were overlain with a scaling grid and were then photographed through a phase contrast microscope. Vesicle diameter sizes were determined from these photographs. Statistics-The data were analyzed using analyses of variance. All data are reported as mean Ϯ S.E.

Characterization of Zucker Rats
Zucker obese rats had a markedly greater (1.7-fold) body weight than their lean counterparts (Table I). Their hearts were slightly (1.3-fold) but significantly enlarged. This increase in mass is likely due to cellular hypertrophy rather than to hyperplasia for the heart (37). Adipose tissue exhibited the most prominent increase in mass as exemplified by the more than 4-fold increase in the amount of epididymal fat. This mass increase is known to be caused by increases in both cell size and cell number (38). There was no difference between obese and lean animals in the quantity of skeletal muscle mass (i.e. hind limb muscles of the lower leg) used in the study (Table I).
The obese Zucker rats exhibited chronically increased blood lipid levels. The circulating FA and triacylglycerols concentrations were 1.9-fold and 6.6-fold higher in the obese Zucker rats. They were also insulin resistant, as indicated by the 4.1-fold higher insulin concentrations but unaltered plasma glucose concentration (Table I). All these results (Table I), are consistent with previous reports concerning this rat model of obesity (39,40).

Giant Vesicle Characterizations in Lean and Obese Zucker Rats
The ratio of plasma membrane protein/total protein in lean and obese animals did not differ in either heart, muscle or adipose tissue (data not shown). The presence of FABPc in the intravesicular lumen is a prerequisite for uptake of FA by giant vesicles, because these proteins act as a FA sink. In heart and muscle, the predominant FABPc isoform is the heart (H)-type FABPc (4). As we have observed previously (4), H-FABPc is more abundant in giant vesicles from heart than from muscle (Table II). In Zucker obese rats the concentrations of heart and muscle H-FABPc in homogenates and giant vesicles were not significantly different from that found in lean heart and muscle.
The diameters of the giant vesicles obtained from heart, skeletal muscle and adipose tissue did not differ in obese and lean animals (Table III). Giant vesicles diameters in heart and muscle correspond closely to those reported previously for Sprague-Dawley rats (4, 41).

Fatty Acid Transport by Metabolically Important Tissues in Lean and Obese Zucker Rats
FA uptake was measured in heart, muscle and adipose tissue from obese and lean rats using giant vesicles, to divorce the uptake of FAs from their subsequent metabolism. Vesicular FA uptake rates were increased in heart (1.8-fold, p Ͻ 0.05, Fig.  1A), skeletal muscle (1.8-fold, p Ͻ 0.05, Fig. 1D) and adipose tissue (1.8-fold, p Ͻ 0.05, Fig. 1G) of the obese Zucker rats compared with their lean littermates.

Content and Localization of FAT/CD36 and FABPpm in Metabolically Important Tissues in Lean and Obese Zucker Rats
In Northern blots from all tissues studied (Fig. 2), the FABPpm transcript was found at 2.4 Kb, which corresponds to observations in rat heart and skeletal muscles in other studies (43). FAT mRNA (Fig. 2) was found at two sizes (2.9 and 2.0 Kb) in all tissues examined. This is also in agreement with other reports (27,42). Changes in both sizes were similar and therefore only the results for the major transcript size are shown (Figs. 2 and 3).
FA Transporters in Heart-FAT mRNA was significantly up-regulated in obese hearts (1.4-fold, p Ͻ 0.05), whereas FABPpm mRNA was somewhat more up-regulated (2.5-fold, p Ͻ 0.05) (Fig. 3). In contrast, there was no difference in the amount of the total available FAT/CD36 and FABPpm transport proteins in lean and obese hearts (p Ͼ 0.05) (Figs. 1B and  4). However, the sarcolemmal content of FAT/CD36 was 1.6fold higher (p Ͻ 0.05) and that of FABPpm 1.5-fold higher (p Ͻ 0.05) in hearts from obese Zucker rats compared with lean rats (Figs. 1C and 4).
FA Transporters in Skeletal Muscle-When compared with muscle of lean rats, there were no significant difference in mRNA levels of FAT and of FABPpm in muscles from obese rats (p Ͼ 0.05, Fig. 3). At the protein level, the total available pool of FAT/CD36 was not changed whereas there was only a small increase (ϩ14%) in FABPpm in the obese rats (p Ͻ 0.05, Figs. 1E and 4). In contrast, the muscle sarcolemmal content of FAT/CD36 was markedly increased (1.8-fold, p Ͻ 0.05, Figs. 1F and 4) in obese rats, whereas there was no significant difference in the sarcolemmal presence of FABPpm in muscles from obese and lean rats (p Ͼ 0.05, Figs. 1F and 4).
FA Transporters in Adipose Tissue-A marked increase in FAT/CD36 mRNA (1.8-fold, p Ͻ 0.05) and in FABPpm mRNA (3-fold, p Ͻ 0.05) was observed in epididymal fat pads of obese Zucker rats (Fig. 3). The amount of total available FAT/CD36 protein was modestly increased (1.3-fold, p Ͻ 0.05, Figs. 1H and 4) in adipose tissue in obese animals. There was no significant difference in the total available FABPpm protein in lean and obese animals (p Ͼ 0.05, Figs. 1H and 4). Plasma membrane contents of FAT/CD36 and FABPpm were increased 1.5fold (p Ͻ 0.05, Figs. 1I and 4) and 1.7-fold (p Ͻ 0.05, Figs. 1I and 4), respectively.
MCT1 in Plasma Membranes-To determine whether the increases in plasma membrane fatty acid transporters were specific, we also measured the MCT1 in plasma membranes of all tissues examined (Fig. 5). Except for adipose tissue plasma  membranes, the MCT1 content in the plasma membranes obtained from heart and muscle did not differ between lean and obese animals. DISCUSSION We investigated the vesicular uptake of FA by metabolically important tissues (heart, skeletal muscle, and adipose tissue) in obese Zucker rats and their lean littermates, using the novel model of giant vesicles in which FA uptake rates are divorced from cellular FA metabolism. We report here for the first time the changes in fatty acid uptake in these metabolically important tissues in obese Zucker rats in relation to the FAT and FABPpm transporters at the mRNA and at the protein levels. Importantly, we examined the total available protein levels of FAT/CD36 and FABPpm, as well as their presence on the plasma membrane.
Our studies demonstrate that, generally, neither the FAT mRNA and FABPpm mRNA abundance nor the total available pool of FAT/CD36 and FABPpm proteins show any consistent relation with vesicular FA transport in a number of tissues. Instead, the increased abundance of FA transporters at the plasma membrane, in heart, muscle, and adipose tissue is most closely associated with changes in vesicular FA uptake in metabolically important tissues of obese Zucker rats. Of the two transport proteins, plasma membrane FAT/CD36 always paralleled the changes in vesicular FA uptake in all tissues, whereas this was observed only for plasma membrane FABPpm in heart and adipose tissue.
Our studies are the first in which FA uptake has been measured in giant vesicles from heart, muscle, and adipose tissue of obese Zucker rats. The differences in FA uptake by heart and muscle in lean and obese animals cannot be explained by differences in cytoplasmic FABPs, because these protein levels were similar in these tissues from obese and lean animals. Similarly, giant vesicle sizes were also similar in these tissues in the lean and obese animals. Thus, in none of the tissues examined can we explain differences in vesicular FA transport in obese and lean animals on the basis of differences in cytoplasmic FABPs or vesicle sizes.
The increased FA transport in obese skeletal muscle is a novel observation, as there are no other studies that have examined FA transport in obese animals in this tissue. The increase in vesicular FA transport in the obese heart parallels the increase in FA utilization observed by Berk et al. (19) in obese Zucker rats. However, they used cardiac myocytes, in which FA uptake and metabolism occur concomitantly, and thus, this preparation does not necessarily reflect solely FA uptake across the plasma membrane. In the same study Berk et al. (19) also observed an increase in adipocyte FA uptake (i.e. FA transport ϩ metabolism) in obese Zucker rats; i.e. a 6.6-fold increase/fat cell or an ϳ3-fold increase when increased cell surface area is taken into account. This 3-fold increase in FA uptake (i.e. FA transport ϩ metabolism) compares favorably with the 2-fold increase in vesicular fatty acid uptake in the present study. Thus, the increases in FA uptake in heart and adipose tissues of obese Zucker rats in our studies using giant vesicles and in the studies of Berk et al. (19) with respiring cardiac myocytes and adipocytes are qualitatively similar.
Although changes in transporter mRNA levels could be associated with the changes in FA uptake by the different tissues, as has been argued by others (19,26,27), it must be stressed that there was not always a clear relationship between FABPpm mRNA or FAT mRNA and the changes in the total available FA transport proteins, their content in the plasma membrane, or FA transport. Except for the small change in skeletal muscle total available FABPpm protein (ϩ14%), there were no changes in the total available FABPpm in any of the other tissues in the obese Zucker rats. There were small changes in total available FAT/CD36 protein in adipose tissue (ϩ30%), but no changes in total FAT/CD36 protein in muscle or heart. We can only speculate about the consistent lack of correlation between mRNA levels and protein levels of FA transporters in obese tissues. It may be that the change in messengers predisposes the obese tissues to react more quickly to changes in nutritional states via a second level of regulation at the translational level. Alternatively, both transcription and translation may be coordinately regulated, whereas there is additional regulation at the level of protein degradation. In any case, the total available protein levels of the FA transporters in the various tissues examined do not offer an adequate explanation for the differences in vesicular FA uptake between lean and obese tissues.
A clear advantage in our studies was the ability to examine in giant vesicles, from a number of tissues, both the FA uptake and the presence of the FA transporters in the plasma membrane of these vesicles. Obesity induced marked increases in FAT/CD36 protein in the sarcolemma of both heart and muscle. Because the total tissue amount of the FAT/CD36 transporter did not change significantly in the heart and muscle, it is possible that in heart and muscle a portion of the intracellular FAT/CD36 pool is permanently relocated to the sarcolemma during obesity. A similar inference may be drawn for adipose tissue, in which the plasma membrane FAT/CD36 was increased considerably more (ϩ46%) than the increase (ϩ26%) in total available FAT/CD36. Alternatively, one or more of the putative the regulatory steps involved in the endocytosis of the fatty acid transporters is impaired.
The novel observation in the present study is that increases in vesicular FA uptake by heart, skeletal muscle, and adipose tissue are due to the increased localization of FAT/CD36 to the plasma membrane. Because the total amount of FAT/CD36 within the cell did not change, it appears that there is an intracellular pool of FAT/CD36 in these tissues, although this suggestion needs to be confirmed in heart and adipose tissue. However, in muscle we have recently found evidence for the existence of an intracellular pool of FAT/CD36 (17). A portion of this pool was recruited to the sarcolemma during muscle contraction (Ͻ30 min), and the relative FAT/CD36 quantity (%) translocated corresponded to a parallel increase (%) in vesicular FA uptake (17). Thus, in muscle the relative contents of sarcolemmal and intracellular FAT/CD36 can be altered, and the translocation mechanism is sensitive to the acute (Ͻ30 min) metabolic needs of the muscle. In obesity the mechanism regulating cellular distribution of FAT/CD36 in muscle is likely to have undergone alterations. Our data suggest that a larger portion of the total FAT/CD36 is maintained at the cell surface at the expense of the intracellular storage compartment. Alterations in transporter redistribution associated with obesity have also been observed for GLUT4 in both muscle (43) and heart (44). In healthy muscles (43) and heart (45) GLUT4 is mobilized by insulin from intracellular stores to the sarcolemma, whereas in obese heart and muscles intracellular GLUT4 translocation by insulin is inhibited (43,45). In contrast, our data show that in obese rats more FAT/CD36 is located at the plasma membrane when compared with lean animals. Taken together, we speculate that the patterns of FAT/CD36 and GLUT4 at the plasma membrane and the in-tracellular compartments appear to be diametrically opposed in obesity.
Another unexpected observation in our study is the difference in the surface content of FABPpm in obese heart and adipose tissue, whereas the total content of total FABPpm in these tissues was not changed. This may indicate that in the heart and adipocytes there are also intracellular pools of FABPpm that can be relocalized to the plasma membrane. In mitochondria-free muscle fractions we have found an intracellular pool of FABPpm, 2 and we are determining whether or not this intracellular FABPpm pool can be translocated to the plasma membrane. Stump et al. (9) have shown that FABPpm is identical to mitochondrial aspartate aminotransferase. Mitochondria are not considered to be part of short term recycling compartments, making it unlikely that this mitochondrial counterpart of FABPpm is involved in the short term translocation of FABPpm to the plasma membrane. However, this mitochondrial FABPpm pool could possibly be involved in a long term relocalization of FABPpm to the plasma membrane. This putative FABPpm relocalization is not observed in muscle in the present study. In this respect it should be noted that tissue-specific differences in transporter recycling are also observed for the glucose transporter family; whereas in the heart both GLUT1 and GLUT4 can be recruited to the sarcolemma by insulin, in skeletal muscle only GLUT4 is translocated by insulin (46).
We cannot determine from the present studies the mechanisms by which FAT/CD36 and FABPpm take fatty acids across the plasma membrane. There has been some speculation (3) and indirect evidence (4) that these transporters may act cooperatively, although definitive evidence for this hypothesis is lacking presently. Increases in vesicular FA uptake in heart, muscle, and adipose tissue reflect qualitatively similar changes in plasma membrane FAT/CD36 in all these tissues. In contrast, plasma membrane FABPpm was increased only in the heart and adipose tissue but was not altered in muscle despite the changes in vesicular FA uptake in all these tissues. Thus, exactly how FAT/CD36 and FABPpm function to facilitate the uptake of FA across the plasma membrane remains unknown. Moreover, whether they are both required is also not known.
A number of parameters were also investigated to ensure that we were examining similar giant vesicles in lean and obese animals and that the data obtained were specific to the fatty acid transport system. The giant vesicle protein recoveries and the giant vesicle diameters were similar in lean and obese animals in the three tissues examined. Also, although the plasma membrane fatty acid transporters were increased in muscle and heart, no increase was observed in the plasma membrane monocarboxylate transporter, MCT1. The increase in adipose tissue plasma membranes MCT1 would therefore seem to be a tissue-specific response to obesity, because protein recoveries did not differ between lean and obese animals in this tissue. Thus, the increases in plasma membrane fatty acid transporters in heart, muscle, and adipose tissue are not to attributable to differences in the giant vesicle protein recoveries, their diameters, or some generalized obesity-induced effect on plasma membrane proteins.
In summary, we have shown that in obese Zucker rats vesicular FA uptake is increased in heart, muscle, and adipose tissue. These alterations in vesicular FA uptake are not attributable to differences in vesicle size or to alterations in the amounts of FA transporters, notably FAT/CD36 and FABPpm, but rather to an increased abundance of these transporters at the plasma membrane, which suggests that the fatty acid 2 J. J. F. P. Luiken and A. Bonen, unpublished data. transporters may be relocated from an intracellular membrane pool to the plasma membrane; or alternatively, their rates of endocytosis are impaired. The use of the giant vesicle preparation, in which we can determine both FA uptake and plasma membrane protein content, was critical to uncovering the mechanism of increased rates of FA transport. The data also establish the importance of FA transporter recruitment to the plasma membrane, first observed in contracting muscle and which now appears to be important in a pathologic state in which there are metabolic changes. Therefore, other pathologic states should also be examined for possible alteration in recruitment of FA transporters. Indeed, because it is increasingly evident that alterations in lipid homeostasis precede the development of insulin resistance, a feature of type 2 diabetes, our new evidence for altered localization of FA transporters might lead to the hypothesis that the cellular machinery regulating subcellular localization of FA transporters might play a pivotal role in the development of insulin resistance.


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Rhein , an Active Metabolite of Diacerein , Suppresses the Interleukin-1-Induced Proteoglycan Degradation in Cultured Rabbit Articular Chondrocytes

Diacerein has proved to be effective in the treatment of osteoarthritis. We investigated the effects of rhein, an active metabolite of diacerein, on the degradation of recombinant human interleukin-1 (rhIL-1 )-induced proteoglycan and matrix metalloproteinases (MMPs) release from rabbit articular chondrocytes. Chondrocytes were treated for 24 h with rhein in the presence of rhIL-1 . Rhein suppressed the rhIL-1 -induced proteoglycan degradation. In addition, rhein decreased the rhIL-1 -induced proMMP-3 production and reduced the MMPs activity. These results suggested that diacerein might have a protective effect on proteoglycan degradation via the suppressive effect of rhein on the production of proMMPs.

Osteoarthritis (OA) is characterized by the destruction of articular cartilage. The extracellular matrix of articular cartilage is composed mainly of collagen and proteoglycan aggregates. In the case of articular diseases such as OA, an alteration of the articular chondrocyte metabolism occurs, resulting in breakdown of the cartilage matrix. Currently, the primary approach in the clinical treatment of OA involves the use of nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics and hyaluronan, which allow symptomatic relief, but provide no apparent disease-modifying effect. In some instances, NSAIDs may even be deleterious; they inhibited the synthesis of proteoglycan, which plays a crucial role in maintaining the function of the cartilage. Therefore, there is a critical need to develop alternative agents that prevent the destruction of cartilage and/ or stimulate its proper repair.
Diacerein has proven to be effective and well tolerated in the long-term treatment of OA, and it is currently under evaluation as a disease modifying OA drug in randomized placebo-controlled clinical trials of patients with OA of the hip and knee (1,2). The efficacy of diacerein has been documented in animal models, including the accelerated canine model of OA (3). Diacerein has a protective effect on granuloma-induced cartilage breakdown by a reduction in the concentrations of proinflammatory cytokines (4). However, the exact mechanisms of therapeutic action of diacerein on OA are not clear. To obtain further insight into the effects of diacerein, monolayer cultures of rabbit articular chondrocytes were used to investigate the effect of rhein, an active metabolite of diacerein (5), on recombinant human IL-1a (rhIL-1a)-induced proteoglycan degradation and the production of promatrix metalloproteinases (proMMPs).
Articular chondrocytes were prepared from the shoulder and knee joints of 3-week-old female Japanese White rabbits (Tokyo Experimental Animal, Tokyo). Chondrocytes isolated by treatment with collagenase were cultured in 100-mm culture dishes at a density of 2.5´10 4 cells / cm 2 in Dulbecco's modified Eagle's medium (DMEM; Life Technologies Inc., Rockville, MD, USA) with 10% fetal bovine serum (BioWhittaker, Walkersville, MD, USA) and antibiotics (100 U/ml penicillin, 100 mg/ ml streptomycin and 250 ng/ ml fungizone ® ; Life Technologies Inc.) in 5% CO2 -95% air at 37°C. Primary cultures maintained in a monolayer were used throughout the study. After the chondrocytes reached confluence, the medium was changed to DMEM with 0.2% lactalbumin hydrolysate (Sigma Chemical, St. Louis, MO, USA) and the antibiotics. Confluent chondrocytes were then incubated with rhein sodium salt (Kyowa Hakko, Tokyo) or naproxen (Sigma) in the presence of rhIL-1a (Genzyme, Cambridge, MA, USA) for 24 h. The concentration of rhIL-1a, at which significant sulfated glycosaminoglycan (S-GAG) release and pro-MMPs production were obtained, was determined in a pre-liminary experiment. Recombinant hIL-1a (0.1 -10 ng/ml) was tested, and the concentration of 1 ng/ ml was selected. Rhein sodium salt was dissolved in distilled water at 3´10 -2 M just before each experiment, and naproxen was dissolved in dimethyl sulfoxide at 1´10 -1 M; they were added to the cell culture at the appropriate concentrations. The final concentrations of distilled water and dimethyl sulfoxide were 0.1% and 0.01%, respectively. The concentrations of rhein were chosen to reflect levels that are below, at or above the established therapeutic anti-inflammatory serum values of rhein after dosing with diacerein described in clinical practice (6). The harvested culture media were stored at -40°C until use. Rhein and naproxen had no effect on chondrocyte viability in this study.
The destruction of articular cartilage results from a failure of chondrocytes to maintain a homeostatic balance between matrix synthesis and degradation. Proinflammatory cytokines such as IL-1, which are produced by monocytes /macrophages, synovial cells or chondrocytes, are thought to play major roles in joint diseases such as OA or rheumatoid arthritis (RA) (7,8). IL-1 has been shown to inhibit extracellular matrix synthesis and stimulate proteoglycan degradation (7). In this communication, IL-1a -induced proteoglycan degradation was assessed by measuring the amount of S-GAG released from chondrocytes into the culture medium. S-GAG was determined using 1,9-dimethylmethylene blue (Aldrich, Milwaukee, WI, USA), as described previously by Chandrasekhar et al. (9). The optical density was then immediately measured at 530 and 590 nm. The concentration of S-GAG was calculated using the standard curve for chondroitin sulfate from shark cartilage (Seikagaku, Tokyo) ranging from 0.3125 to 20 mM. When chondrocytes were treated with rhIL-1a for 24 h, the S-GAG levels in the conditioned medium were significantly higher than those of the untreated control (Table 1). Rhein (10 and 30 mM) significantly inhibited the rhIL-1a-induced S-GAG release (28.9% inhibition at 10 mM and 27.8% inhibition at 30 mM). Naproxen (1 mM) had no effect. The stimulatory effect of IL-1 on the proteoglycan degradation and its inhibitory effect on the synthesis of the new proteoglycan were not inhibited by NSAIDs (10). It is well known that many proteinases, such as MMPs, cysteine proteinases and serine proteinases, and reactive oxygen species (ROS), such as hydroxyl radicals, hydrogen peroxide, superoxide and nitric oxide (NO), which are found in connective tissues and inflammatory cells, can degrade cartilage matrix components. Numerous studies have demonstrated that MMPs are most frequently implicated in the destruction of articular cartilage in arthritic diseases. Indeed, the level of MMPs, tissue inhibitors of MMPs (TIMPs), and proinflammatory cytokines in the serum and the synovial fluid of patients with OA or RA have been shown to significantly increase (7). Furthermore, proinflammatory cytokines induce and/ or augment the production of proMMPs.
We determined the amount of proMMP-3 in the conditioned medium using the MMP-3 enzyme immunoassay (EIA) system (Amersham Pharmacia Biotech, Tokyo) according to the manufacturer's instructions. When chondrocytes were treated with rhIL-1a for 24 h, the production of proMMP-3 increased by about 34-fold as compared with that of the untreated control (Table 2). Rhein (10 and 30 m M) significantly inhibited the production of proMMP-3 (32.0% inhibition at 10 mM and 56.7% inhibition at 30 mM). Naproxen had no effect.
The activities of MMPs are controlled at several levels including proenzyme activation and regulation by the specific MMPs inhibitors, TIMPs. We investigated whether the effect of rhein resulted in the decrease in MMPs activity. The apparent activity of the MMPs in the conditioned medium was determined using fluorescein isothiocyanate  Confluent chondrocytes were treated for 24 h with drugs in the presence of rhIL-1a , and then the proMMP-3 concentration in the conditioned medium was assayed using the EIA system. Each value represents the mean ± S.D. for 5 independent experiments. **P<0.01 and ***P<0.001, significantly different from the rhIL-1a -treated control group by Dunnett's test. ### P<0.001, significantly different from the rhIL-1a -treated control group by Aspin-Welch's test.
(FITC)-labeled casein as a substrate according to the method of Twining (11) with slight modifications of the microassay. Before the assay, proMMPs in the conditioned medium were activated by L-1-p-tosylamido-2-phenylethyl chloromethyl ketone treated-trypsin followed by the addition of a four-molar excess of soybean trypsin inhibitor. The enzyme activity that digests 1 m g of casein for 1 h at 37°C was expressed as 1 U. Recombinant hIL-1a significantly augmented the caseinolytic activity compared to the untreated control (Table 3). Rhein (10 and 30 mM) significantly suppressed the rhIL-1a-enhanced caseinolytic activity (14.3% inhibition at 10 m M and 37.7% inhibition at 30 mM). Naproxen had no effect. Next, the MMP-9 activity was determined using the MMP-9 activity assay system (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The MMP-9 activity also significantly increased as compared with that of the untreated control (Table 3). Rhein (30 mM) significantly inhibited the MMP-9 activity (11.8% inhibition). Naproxen had no effect. The MMPs are well known to degrade proteoglycan. In this communication, we have shown that rhein suppresses the rhIL-1a-induced S-GAG release along with a decrease in production of proMMP-3. This is the first evidence that rhein down-regulates the proMMP-3 production in rabbit articular chondrocytes that was determined with an enzyme immunoassay system. The proMMPs suppression of rhein may be involved in the inhibition of S-GAG release from chondrocytes.
The mechanism by which rhein inhibits the proteoglycan degradation and proMMPs production is not fully understood at present. Cruz et al. (12) reported that the inhibition of the activator protein-1 activity might serve as the mechanism of diacerein that inhibits collagenase expression in bovine articular chondrocytes, but the action of rhein is not specified. On the other hand, it has been suggested that blocking the ROS production or decreasing the ROS levels reduces the IL-1-induced collagenase expression in the chondrocytes (13). Rhein is known to reduce the production of superoxide (14) and NO (15). Taken together, it is likely that rhein modulates the proMMPs production and activity via interference with multiple signaling pathways and thus protects the cartilage. Rhein may have a direct protective role on chondrocytes, or an indirect effect through inhibition of the proMMPs production. Further study is necessary to clarify the involvement of rhein induced proMMPs suppression in proteoglycan degradation.
To conclude, rhein, an active metabolite of diacerein, inhibits the destruction of articular cartilage induced by rhIL-1a. These findings may be related to the suppressive effect of rhein on the proMMPs production. Confluent chondrocytes were treated for 24 h with drugs in the presence of rhIL-1a, and then the caseinolytic or MMP-9 activity in the conditioned medium was determined using FITC-labeled casein or the MMP-9 activity assay system. Each value represents the mean ± S.D. for five independent experiments. *P<0.05 and **P<0.01, significantly different from the rhIL-1a -treated control group by Dunnett's test. ## P<0.01 and ### P<0.001, significantly different from the rhIL-1a -treated control group by Aspin-Welch's test. NT: not tested.


extfieldsofstudy: Biology Medicine Physics

text:
On the statistical mechanics of prion diseases

We simulate a two-dimensional, lattice based, protein-level statistical mechanical model for prion diseases (e.g., Mad Cow disease) with concommitant prion protein misfolding and aggregation. Our simulations lead us to the hypothesis that the observed broad incubation time distribution in epidemiological data reflect fluctuation dominated growth seeded by a few nanometer scale aggregates, while much narrower incubation time distributions for innoculated lab animals arise from statistical self averaging. We model `species barriers' to prion infection and assess a related treatment protocol.

Transmissible neurodegenerative prion diseases, such as Mad-Cow Disease (BSE) and Creutzfeldt-Jakob Disease (CJD) in humans, increasingly represent a serious public health threat [1]. Prusiner and collaborators have shown that the infectious agent (prion) in these diseases consists of a quantity of a misfolded form (PrP Sc ) of the ∼200 amino acid PrP C protein which is expressed ubiquitously in mammalian neurons [2]. The PrP C proteins normally reside on neuron surfaces [4], and the more hydrophobic PrP Sc forms tend to aggregate: large (micron scale) PrP Sc amyloid plaques are a common post-mortem feature of brain tissues, and fibrils are observed in in vitro experiments. Nucleic acid free propagation demands that PrP Sc autocatalyze their own formation by helping to convert more PrP c into PrP Sc [2,3]. Given that mammalian PrP c differ by only 5-10% in amino acid composition and that variant CJD correlated with BSE has been observed in England, the efficacy of "species barriers" in limiting transmission is of considerable interest.
Several facts suggest that prion disease may be driven less by complex biology (as in Alzheimers' disease) than by physico-chemical processes, including: (1) The "universal character" of sporadic CJD, observed globally without evident spatio-temporal clustering at an annual background death rate of 0.5-1.5/million and mean onset age of about 63 years [6]. (2) Highly reproducible incubation time vs. infectious dose distributions observed in laboratory animal studies, with a power law relation between mean incubation time and dose concentration D [7]. (3) The close temporal proximity of disease onset and death. (4) PrP Sc may be grown in vitro without biological processing and is toxic to neurons, though not yet demonstrably infectious [5]. The following question is thus raised: is the incubation time of prion disease dominated by a fundamental physico-chemical nucleation and growth of PrP Sc protein aggregates?
We present simulation results for a simple twodimensional, lattice based statistical mechanical model for prion disease based at the protein level. It is all but hopeless to develop an atomic level simulation capable of spanning the 21 temporal orders of magnitude between picosecond scale intra-protein motion and the potentially decades long incubation times of large prion aggregates. Our simulation is a bridge between the short distance and time scales covered in individual protein models and the long time, macroscopic realm of chemical kinetics. We use our simulations to identify the protectorate of principles necessary to describe the aforementioned universal features of prion disease. Specifically, we find: (1) Concommitant PrP Sc autocatalysis and stable aggregation can explain the long disease incubation times, and the difference between sporadic (unseeded) and infectious (seeded) onset. This principle is consistent with data for yeast prions [10], is closely related to the earlier nucleation theory of Lansbury [8], and avoids the need to fine tune model parameters with separated autocatalysis and aggregation [9]. (2) The distributions of disease incubation times (well defined onset edge, broad fluctuation driven spectrum for dilute prion doses, narrow short time shifted distribution for more concentrated prion doses) stongly suggest a central role for growth from a minimally nanometer scale seed (with order 10 PrP Sc proteins). With these assumptions we obtain a one parameter fit to the BSE incubation time data, an order of magnitude correct estimate of the mean incubation time for BSE and infectious CJD, and we argue that self-averaging explains a narrowing of incubation time distributions observed in the laboratory [7,11]. Finally, by considering both sticking and interconversion rate differences for prions of different species, we provide a theoretical underpinning to data for the species barrier efficacy and to proposed treatments based upon coating of incipient prion plaques.
Our assumption of two-dimensionality is consistent with the observation that PrP c resides on neuron surfaces and autocatalysis and aggregation must certainly initiate there. The lattice structure should be irrelevant at long times. By assuming a triangular lattice we minimize the anisotropy (which appears first in rank six tensors for hexagonal lattices). This simulation yields the crucial qualitative features necessary to specify the "prion protectorate." Our resulting prion aggregates are essentially amorphous, rather than quasi-one-dimensional filaments as found in some in vitro experiments. We shall discuss an anisotropic simulation yielding fibrils elsewhere.
Our model is based upon the energy landscape schematized in Fig. 1. Individual lattice cells take on three values: unoccupied (implicitly filled with water), occupied by a PrP c protein, or occupied by a PrP Sc protein. At the beginning of each simulation, we choose a monomer PrP c configuration distributed randomly, with a number (0-4) of PrP Sc seeds located randomly as well.
In the spirit of cellular and molecular automata, we eschew an explicit Hamiltonian based molecular dynamics in favor of the following updating rules, specified in time units of one full lattice sweep (d is the nearest neighbor coordination number): (1) Identify each occupied cell as PrP C or PrP Sc by checking its number of nearest neighbors.
(2) For each PrP C , move one step in a random direction if that adjoining site is unoccupied.
(3) Identify aggregates (size N P ) by PrP Sc presence; move them per Rule (2) with probability 1/ N p . (4) For d = 0, 1 the protein remains PrP C . (5) For d = 2, the protein is PrP C or PrP Sc with equal probability (6) For d ≥ 3 the protein is PrP Sc .
Rules (1-3) ensure diffusive motion of PrP C monomers and PrP Sc aggregates. Rules (4-6) specify protein interactions and autocatalysis, and reflect the relative hydrophobicity of PrP Sc . To assure the biologically plausible condition of constant average PrP C concentration, at the start of a new sweep we randomly place a new PrP C for each lost in the previous update.
This procedure is repeated for up to millions of full sweeps through the lattice. Note that a sweep defines the minimum time scale for conversion of PrP C to PrP Sc , which is presumably of order a second or less in real time. We work with maximum lattice sizes of N = 4 × 10 4 . Practically, we are able to run at areal monomer concentrations c ≥ 0.1%, which are likely to be about three orders of magnitude larger than in the brain.
We run our seeded simulations repeatedly following the largest nano-aggregates until they reach a size of up to 0.4% of the lattice. Each seed consists of 10 PrP Sc monomers [12] For a given aggregate size, we have studied the properties of the distribution of lifetimes to reach that size. For a final aggregate size of A=0.2%N (80 monomers) we display the corresponding distributions P D in Fig. 2(a) for several values of c and D = 1. P D displays greater fluctuations for small c, and increasing c shifts P D to short times. Fluctuations dominate the growth from the small seed aggregate. For larger A we find that the onset time grows sublinearly in A, while the distribution width grows weakly with A. To within fluctuations, the P D curves for different c collapse when scaled by the mean time t m . (The curves broaden somewhat for D = 2, 3, 4.) This implies the scaling law In the range of concentrations studied, we can fit t m Taken together, these data lead us to a conjecture of asymptotic compression of P D : for c → 0, an increasingly large range of A will have essentially the same scaled shape, because the time t 2 it takes to go from A to say, 2A will be much smaller than t m for going from 10 → A.
We believe that other processes, such as protease attack or competition between PrP Sc -PrP Sc and PrP Scneuron binding will limit prion aggregation on the neural surface and cause fissioning and spreading of nanoaggregates to other neurons. Such fission is necessary for exponential growth of infectious agents, as noted previously [14]. At the order-of-magnitude level t 2 sets the doubling time for disease spread, assuming that intercellular diffusion rates exceed intra-cellular ones. Given t 2 << t m (at biological c values) both in our fission hypothesis and experiment, slow growth fluctuations for small seed aggregates can dominate the overall incubation time for disease. For example, in hamster experiments the first observable levels of PrP Sc in brain tissues occur at about 90 days, with symptom onset at 120 days, and a doubling time of 2 days [13]. Given the conjectured asymptotic compression, the incubation time distribution will be relatively insensitive to a wide range of nano-aggregate sizes for fission parents or progeny.
We stress several points of agreement with observation and laboratory data. First, we always find death (runaway growth of a killer aggregate) in the presence of infection, in agreement with experiment and the aggregate nucleation hypothesis for prion disease. Second, our rough scaling of t m with D agrees qualitatively with the extraordinarily reproducible dose vs. incubation time data of Prusiner and collaborators for prion infection in laboratory mice, for which τ I ∼ D −.62 . Third, our lifetimes are in order of magnitude agreement with observed BSE incubation times and new variant CJD death ages, which we can prove by exploiting our concentration scaling. For a single seed, at our lowest concentration of 0.1% our distribution peaks at about 10 4 sweeps. Assuming a literature estimate of concentration of in vivo prions in solution of 100 nanomole/liter [15] and a protein diame-ter of order one nanometer suggests a dimensionless areal concentration of about 10 −3 %. With the above concentration scaling this gives an incubation time of about 10 8 sweeps. If we take the 5 year mean incubation time for BSE this implies our basic misfolding time (one sweep) is about one second, which is reasonable at the order of magnitude level. Finally, by simply scaling time for A = 80 by the mean time t m , we achieve an exceptionally good fit (A) of the inferred incubation time distribution for BSE in the United Kingdom (for cattle born in 1987) [16] to our scaling curve as shown in Fig. 2(b). This supports our hypothesis that runaway autocatalytic aggregation seeded by a few prion nano-aggregates triggers the disease. This is very plausible since (i) infection is believed to have come from rendering plant offal derived from many animals after considerable processing,leading to substantial dilution, and (ii) prion transmission by oral consumption is estimated to be less efficient than innoculation by a factor of 10 9 .
Sporadic CJD develops in the absence of any infection or genetic predisposition to prion disease. We have studied the lifetime distributions in this case and find that they are very different in shape than the seeded runs, being much broader and dimer fluctuation dominated (peaks scale as c −2 ). For our estimated biological concentration of c = 10 −3 %, this could give a mean onset time as large as 10 3 years. Assuming a constant height tail from the minimum single seed onset to the mean human lifespan taken with the 1 part in 10 6 odds of dying from sporadic CJD gives an estimate of a total death probability of a part in 10 5 up to this mean on-set time. This is consistent with our simulations, but not resolvable currently.
In contrast to the breadth of our single seed lifetime distributions, in the event of dosing by a greater number of seeds, self-averaging will narrow the distribution, collapsing it towards the onset (shorter times). Assuming growth proceeds independently for different seeds (which is quite reasonable for the low concentrations in living organisms) distributions would narrow by √ D. Several in vivo experiments show distribution time narrowing with increased doseage, but are not amenable to quantitative analysis at this time [7,11].
We turn now to the species barrier. To simulate interspecies transmission we replace the integer-valued coordination, the key parameter which determined the stability of different protein conformations in the earlier studies, by a new variable x = N + N ′ × P for PrP C and x ′ = N ′ + N × P ′ , for PrP C′ , where N is the number of PrP C,Sc neighbors and N ′ the number of PrP C,Sc′ neighbors. The parameters P and P ′ (chosen less than unity) provide a measure of the reduced effectiveness of interspecies conversion. To deal with the continuous variable x, rather than the integer coordination used earlier, we represent the PrP C to PrP Sc conversion probability f (x) with a similar definition for PrP C′ →PrP Sc′ conversion. A separate parameter describes physisorption to an aggregate of alien prions. We allow a PrP C,Sc cluster of mass M (M = 1 being a monomer) to desorb from an alien prion aggregate with probability q K / √ M , where q is the physisorption parameter, and K is the number of units in the cluster adjacent to the alien aggregate. We define q ′ for PrP C,Sc′ similarly. Thus, we describe species barriers in terms of the two parameters affecting conversion rates (P, P ′ ) and physisorption (q, q ′ ). Making P = P ′ and q = q ′ allows for an asymmetry in infectivity, and we can model one of the most striking aspects of the species barrier, namely its asymmetry (e.g. mice infect hamsters well, hamsters infect mice poorly).
We have carried out several simulations relevant to the species barrier. First, we have placed alien prion 'walls' on a boundary, representing a single large aggregate from another species. With suitable choice of parameters, this leads to the asymmetric species barrier between two species. Namely, disease onset in one species (with large aggregates) and only sub-critical PrP Sc concentration near the wall in the other. Recent experimental data has also shown the build up of such sub-critical PrP Sc concentration in the asymmetric interspecies transmission [17].
Second, suppose the asymmetry is such that P << P ′ . In this case, PrP Sc′ is more favorably formed from PrP C′ in the presence of a seed aggregate of PrP Sc than PrP Sc is formed from PrP C in the presence of a PrP Sc′ seed. Thus, by injecting a large enough initial dose of alien PrP C′ proteins into the organism, it should be possible for PrP C′ to compete favorably with PrP C for aggregation, and thus extend the incubation time. Such protocols have been tested experimentally: (i) Experiments with the coating dye molecule Congo Red [18] reveal a surprising non-monotonic dose vs. lifetime relation: small Congo Red concentrations yield a reduced time for incubation, while larger Congo red concentrations significantly extend the incubation time. (ii) In vitro experiments show that the above coating scenario with alien prions works [19] when the initial PrP C′ concentration c ′ > c.
We find both of these experimental features in our simulation as shown in Fig. 3. For c ′ /c < 2, the incubation time is slightly shortened, while for c ′ /c > 2 the incubation time is increased. We find that the lifetime shortening arises because once a few PrP C′ aggregate, they partially block the motion of adjacent PrP C proteins, enhancing the misfolding probability of the latter. However, further PrP C misfolding is significantly reduced once PrP Sc′ coats the seed.
A crucial test of our theory would be to monitor, in vitro, the lifetime distributions of normal prion proteins seeded by small aggregates as a function of both c and D to look for the scaling behavior. If it is not possible to easily achieve D = 1, 2, presumably the distribution of Fig. 2(b) could be estimated by extrapolation from successively diluted PrP Sc doses. This would also test the extent to which the self-averaging applies. If our single seed hypothesis is correct, it is important to assess how passage occurs upon infection.
Theoretically, it is important to extend our model by including internal degrees of freedom for the proteins which can provide an orientational dependence to the aggregation, allowing for linear fibril formation as opposed to amorphous aggregates. This connects naturally with simulations on individual proteins, and also allows for the study of different protectorates for collective protein phenomena.


extfieldsofstudy: Biology Medicine

text:
Akt Is Activated in Response to an Apoptotic Signal*

Akt is a serine-threonine kinase known to exert antiapoptotic effects through several downstream targets. Akt is cleaved during mitochondrial-mediated apoptosis in a caspase-dependent manner. The reason for this is not clear, however, because Akt has not been demonstrated to be activated in response to mitochondrial apoptotic stimuli. Accordingly, we explored whether the well described mitochondrial apoptotic stimuli stauro-sporine (STS) and etoposide activate Akt and whether such activation impacts apoptosis. Both STS and etoposide activated Akt in NIH 3T3 cells, maximally at 8 and 2 h, respectively, preceding the onset of apoptosis and poly(ADP-ribose) polymerase cleavage. The overexpression of Akt delayed STS-induced apoptosis with an even more pronounced delay observed with overexpression of constitutively active Akt. Akt activation by proapoptotic stimuli lay upstream of mitochondria, because neither caspase inhibitors nor overexpression of Bcl-2 or Bcl-x L could prevent it. Activation depended on phos- phatidylinositol 3-kinase activity, however. Conversely, inhibition of phosphatidylinositol 3-kinase with wortmannin sensitized cells to apoptosis initiated by STS. These data demonstrate that mitochondrial apoptotic stimuli also activate Akt and such activation modulates apoptosis in this setting. membranes dilution 20 phosphatidylinositol 3-kinase; NF- nuclear staurosporine; PARP, polymerase; synthase pendent

Apoptosis is an important cellular response to irreparable damage from exposure to external stimuli or failure to receive survival signals (1). Common to the process is the activation of caspases, aspartic acid-directed proteases (2). For apoptosis to effectively occur, survival pathways must be inhibited or fail to be activated. In this regard, Akt (also termed protein kinase B or PKB) has been found to be cleaved in a caspase-dependent manner during Jurkat cell apoptosis mediated by Fas ligation, UV irradiation, or etoposide (ETOP) 1 (3).
Akt is phosphorylated and activated by phosphatidylinositol 3-kinase (PI3K) in response to mitogens (especially Ras signaling) and adhesion (4,5). It has been demonstrated that signaling through PI3K/Akt is necessary for transformation by acti-vated mutant Ras (6,7). Akt delivers an antiapoptotic survival signal by several mechanisms including the phosphorylation and inactivation of Bad (8) and caspase-9 (9) as well as stimulation of NF-B activity through the activation of IKK␣ (10 -12) and inactivation of forkhead transcription factor pathways (13,14). There is also evidence that the transforming potential of Akt depends on the protein kinase mTOR (15). Constitutive activation of the PI3K/Akt pathway has been observed in several human cancers including ovarian (16), breast (17,18) and prostate cancers (19). Furthermore, the inhibition of Akt signaling can induce apoptosis in some human cancer cell lines (16,20).
Importantly, it also seems that Akt signaling can affect the response to chemotherapeutic agents. Inhibition of Akt phosphorylation is seen with topotecan, and the re-expression of phosphorylated Akt renders human lung cancer cells less sensitive to topotecan treatment (21). Similarly, ovarian cancer cells overexpressing constitutively activated Akt are resistant to paclitaxel (22).
The observation that cleavage of Akt occurs during apoptosis (3) suggests that either a level of baseline Akt signaling is vital for cell survival or that Akt activation occurs during apoptosis and acts as a "brake" on the process. Consequently, we have studied the behavior of Akt during apoptosis and indeed find activation of Akt and show that this has functional importance.

EXPERIMENTAL PROCEDURES
Materials and Cell Culture-STS, ETOP, and the PI3K inhibitor wortmannin were from Sigma. STS and ETOP were used at concentrations of 0.1-1.0 and 10 -100 M, respectively. zVAD, a pan-caspase inhibitor, was from Enzyme Systems Products. Wortmannin and zVAD were dissolved in Me 2 SO. Wortmannin and zVAD were used at final concentrations of 100 and 50 M, respectively. NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum (Life Technologies, Inc.), streptomycin (100 g/ml), penicillin (100 units/ml), and 2 mM glutamine at 37°C in 95% air/5% CO 2 .
Akt Activity Assay-Akt activity was measured in vitro using histone H2B as the substrate. After protein isolation from total cell lysate as described above, 200 g of total protein was then incubated with anti-Akt antibody immobilized on protein-Sepharose A beads (1:100; New England Biolabs) with gentle rocking overnight at 4°C. The beads were washed three times with the lysate buffer and once with the kinase buffer (25 mM Tris, 5 mM ␤-glycerophosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, and 10 mM MgCl 2 ) and resuspended in 50 l of 1ϫ kinase buffer supplemented with 2 g of histone H2B and 1 mg/ml bovine serum albumin. Akt activity was then determined by the addition of 200 M [ 32 P]ATP (1000 cpm/pmol) incubated for 30 min at 30°C. The reaction was terminated with the addition of 3ϫ SDS sample buffer (187.5 mM Tris-HCl (pH 6.8), 6% w/v SDS, 30% glycerol, 150 mM dithiothreitol, and 0.3% w/v bromphenol blue), boiled for 5 min, and analyzed by SDS-polyacrylamide gel electrophoresis. Signals were detected by exposing the gel to x-ray film (X-OMAT, Kodak).
Cell Death Assay and Activation of Apoptosis-Quantitative cell death was measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay as published previously (23). Activation of the apoptotic cascade was measured by immunoblotting of PARP cleavage as outlined above and as published previously (23). DNA fragmentation after exposure to STS or ETOP was assayed as published previously (23).
Transfection-Constructs were made by subcloning HA-tagged bovine wild-type Akt, a constitutively active Akt (Akt-DD), human Bcl-2, and Bcl-x L into the pBabe retroviral vector. Plasmid DNA was isolated and used to generate the retrovirus. High titers of retrovirus were generated using phoenix packing cells. Briefly, 5 ϫ 10 6 phoenix cells were seeded in a 10-cm plate overnight before transfection with 15 g of retroviral vector for 56 h. The virus-containing medium was harvested after 48 and 56 h. The combined medium was filtered through a 0.45-m filter. After the addition of 10 g/ml polybrene (Sigma), the medium was used to infect NIH 3T3 cells for 24 h. The infected cells were then selected with puromycin at 1.5 g/ml for 48 h. The infection rate was more than 90% as determined by infecting NIH 3T3 cells with the pBabe/LacZ retrovirus. Protein expression was confirmed with Western blot as described above.

Mitochondrial Apoptotic Stimuli Lead to Akt Activation-
Recently it was shown that the activation of Akt mediates survival of cells in various death settings. Forced expression of Akt protects neuronal cells from apoptosis induced by withdrawal of the survival factors insulin-like growth factor-1 or nerve growth factor (24,25) and protects from interleukin-3 withdrawal-induced apoptosis in hematopoietic cells (26). The activation of Akt also protects cells from apoptosis induced by detachment from extracellular matrix and c-myc overexpression (7,27). The common feature of these apoptotic processes, all inhibited by the forced expression of Akt, is that they are executed primarily through the mitochondrial apoptotic pathway.
NF-B, which strongly protects against death receptor-induced apoptosis, is activated by TNF␣ ligation of death receptors in concert with the initiation of the death signal (12). The NF-B activation observed in this setting is through the PI3K/ Akt pathway (12). There is also evidence of NF-B activation after ETOP treatment associated with an increase in A1, a Bcl-2-like protein (28). Given this and the known ability of forcibly expressed Akt to inhibit mitochondrial apoptosis, we sought to determine whether well established mitochondrial apoptotic stimuli would activate Akt. To address this, NIH 3T3 cells were treated initially with STS, a potent protein kinase inhibitor that induces apoptosis by directly stimulating cytochrome c release from mitochondria (29). STS exposure led to caspase activation and cell death as predicted (Fig. 1A, upper panel). PARP cleavage was demonstrable at 8 h, correlating well with the exponential increase in cell death observed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining beginning by 8 h. Interestingly, besides in-ducing apoptosis, however, STS also led to Akt activation detected by increasing levels of Ser-473 phosphorylation beginning by 2 h and maximally at 8 -12 h (Fig. 1A). Three events are required for Akt activation: recruitment to the cell membrane through the N-terminal PH-domain, Thr-308 phosphorylation in the kinase domain, and Ser-473 phosphorylation in the C-terminal tail (30). Although phosphorylation on Ser-473 always correlates with in vivo Akt activation, to ensure that increases in phospho-Akt represented increases in Akt activity the phosphorylation of a histone H2B target protein in the presence of immunoprecipitated Akt was measured. Consistent with the phospho-Akt data, marked histone H2B phosphorylation was observed after 8 h of STS treatment (Fig. 1B). As expected, the positive control platelet-derived growth factor also led to marked histone H2B phosphorylation.
Because STS is a potent inhibitor of many protein kinases, it could activate Akt by the blockade of an Akt-directed protein kinase that delivers a negative phosphorylation signal. To address whether Akt activation in an apoptotic process induced by STS is unique, we examined ETOP-induced apoptosis, which is also known to be executed through the mitochondrial apoptotic pathway (31,32). Akt phosphorylation was observed 2 h after the addition of ETOP to NIH 3T3 cells (Fig. 2, upper panel). The kinetics are different, however, in that maximal Akt phosphorylation induced by ETOP occurred at 2-4 h, which contrasts with the 8-h time point maximum seen with STS. Phosphorylation correlated with activity, as revealed by Akt-dependent phosphorylation on Ser-21 of glycogen synthase kinase 3-␣ (GSK3␣), a known specific Akt target (33) (Fig. 2,  lower panel). These data indicate that Akt activation, usually associated with the inhibition of cytochrome c release (34), occurs in the presence of apoptosis induced by agents that require cytochrome c release for their actions (32).
Akt Activation Protects against Mitochondrial Apoptotic Cell Death-To determine whether Akt activation is functional in mitochondrial apoptotic cell death, we wished to determine whether the activation of Akt affected cellular execution in these settings. HA-tagged bovine Akt, either in its native form (HA-Akt) or in a constitutively active form (HA-Akt-DD) generated by the substitution of Thr-308 and Ser-473 with an aspartic acid (D), was successfully overexpressed in NIH 3T3 cells by retroviral infection (Fig. 3A). The apoptosis, PARP cleavage, and phosphorylation of both endogenous Akt and HA-Akt were examined in NIH 3T3 cells infected with pBabe, pBabe/HA-Akt, and pBabe/HA-Akt-DD in response to STS stimulation. As expected, the treatment of vector retrovirusinfected (pBabe) cells with STS led to Akt phosphorylation, again maximal at 8 -12 h, and PARP cleavage was again observed by 8 h (Fig. 3B). Overexpression of native HA-Akt led to the appearance of two phospho-Akt bands after STS treatment, with the slower migrating band being the HA-tagged overexpressed protein. PARP cleavage was clearly delayed by the overexpression of Akt, now occurring maximally between 8 and 12 h (Fig. 3B). The phospho-specific anti-Ser-473 antibody, 4E2, again detects the activation of endogenous Akt in NIH 3T3 cells infected with pBabe/HA-Akt. The antibody also detects the DD form of Akt with Ser-473 substituted by a D residue even in untreated cells, indicating that the antibody recognizes the negative charge at residue 473 rather than the phosphorylated Ser-473 itself. However, 4E2 does have a much higher affinity for this region when Ser-473 is phosphorylated rather than replaced with a D substitution (data not shown). Overexpression of constitutively active Akt (HA-Akt-DD) was even more effective than overexpression of wild-type Akt at delaying PARP cleavage, with cleavage not observed until 24 h in this condition (Fig. 3B). These data indicate that the activa-tion of Akt in response to STS may have functional significance and suggests that Akt may act as a brake on apoptotic processes. This is consistent with the notion that survival functions are also initiated along with the activation of an apoptotic process. It should be noted that ultimately, by 24 h PARP cleavage is complete under all conditions, indicating that Akt activation functions to delay rather than diminish apoptosis.
We reasoned that if the activation of Akt functions as a brake to slow down apoptosis, inhibition of Akt activation might sensitize cells to apoptosis. NIH 3T3 cells were treated with or without the PI3K inhibitor wortmannin together with 0.1 M STS or 10 M ETOP. DNA fragmentation was observed at 12 h, with co-incubation with 100 nM wortmannin rendering it more prominent (Fig. 4A). Similarly, PARP cleavage was also observed at 12 h, again more prominently with wortmannin (Fig.  4B). Quantification of cell death with terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling confirmed that 19.4% of STS-treated cells were dead at 12 h, and this was increased to 35% with wortmannin co-incubation (Fig. 4, B and  C). Similar data were observed with ETOP (data not shown). These results indicate that wortmannin-sensitive Akt activation in response to STS or ETOP indeed does have functional significance and may delay apoptosis.

STS-induced Akt Activation Takes Place Upstream of Mitochondria and
Requires PI3K Activity-One can distinguish whether an apoptotic process is initiated through death receptors or mitochondria by determination of the relationship between cytochrome c release from mitochondria and caspase activation. Cytochrome c release from mitochondria depends on Bid cleavage by caspase-8 in the death receptor apoptotic pathway and is independent of caspase activity in the mitochondrial apoptotic pathway (29). Consequently, we sought to determine whether the inhibition of caspase activity would affect Akt activation in response to STS. zVAD, a broad spectrum caspase inhibitor, inhibited PARP cleavage but was without effect on Akt phosphorylation after 8 and 12 h of STS treatment of NIH (PDGF) at 100 ng/ml for 30 min after serum starvation for 5 h. Cell lysate was prepared, and Akt was immunoprecipitated with an anti-Akt antibody as described under "Experimental Procedures." The precipitated Akt was used to phosphorylate histone H2B in vitro and was detected by Western blot using an anti-Akt antibody.
3T3 cells (Fig. 5A). Subsequently we wished to determine whether STS-induced Akt activation depends on PI3-kinase activity. NIH 3T3 cells were treated with STS with or without 100 nM wortmannin. Wortmannin completely abolished STSinduced Akt activation (Fig. 5B), indicating the dependence on PI3-kinase activity for STS-induced Akt activation.
To demonstrate firmly that Akt activation in response to apoptotic stimuli occurs upstream of mitochondria, we wished to address whether inhibition of cytochrome c release would affect STS-induced Akt activation. The expression of either Bcl-2 or Bcl-x L is known to maintain the integrity of mitochondria and to prevent cytochrome c release in response to diverse apoptotic stimuli (35). NIH 3T3 cells were infected with pBabe, pBabe/Bcl-2, or pBabe/Bcl-x L . Successful overexpression of human Bcl-2 and Bcl-x L is shown in Fig. 6A. STS-and ETOPinduced apoptosis was abrogated in cells overexpressing either Bcl-2 or Bcl-x L but not pBabe (data not shown). The overexpression of Bcl-2 or Bcl-x L has no effect on STS-induced Akt phosphorylation, however (Fig. 6B). This indicates that STS activation of Akt does not require mitochondrial involvement and indicates firmly that the activation of Akt seen in response to this apoptotic agent occurs upstream of mitochondria. DISCUSSION Proliferation and death (apoptosis) are two sides of the same coin. Although their functions are opposed, they are tightly connected. Deregulation of the oncogenes myc, ElA, or E2F drives cell cycle progression but may also promote apoptosis in certain conditions (36). The mitotic and proapoptotic properties of c-myc require intact N-terminal transcriptional, DNA binding, and dimerization domains (37,38). However, transcription-defective c-mycS is also capable of promoting apoptosis as well as retaining the ability to promote cell proliferation (39). The functions of E1A in promoting proliferation and apoptosis are also linked and were both mapped to the N-terminal region that is involved in binding pocket proteins such as Rb (retinoblastoma protein), which is essential in the regulation of cell cycle progression (36,40). Mutations in E2F that prevent binding to Rb accelerate both cell cycle progression and promote apoptosis (41).
Conversely, apoptotic stimuli may activate counterbalancing survival functions. The binding of TNF␣ to TNF receptor-1 activates caspase-8 and NF-B in a TNF receptor-associated death domain protein-dependent manner (42,43). The activa-FIG. 2. Activation of Akt by ETOP in NIH 3T3 cells. NIH 3T3 cells were treated with either platelet-derived growth factor (PDGF) as described in the Fig. 1 legend or with ETOP (100 M) for the times indicated. Akt activation was determined by Western blot using a phospho-specific anti-Akt antibody (upper panel) or using a phospho-specific anti-GSK3␣ antibody (lower panel).

FIG. 3. STS-mediated Akt activation delays apoptosis.
A, NIH 3T3 cells were infected with retroviruses of pBabe (vector), HA-Akt, or constitutively active HA-Akt-DD as indicated. Akt expression was determined by Western blot using an anti-HA antibody (12CA5). B, NIH 3T3 cells infected with pBabe, HA-Akt, and HA-Akt-DD were treated with STS at 1 M for the times indicated. Retrovirus pBabe-infected NIH 3T3 cells were also treated with platelet-derived growth factor (PDGF) as outlined in the Fig. 1 legend to provide a positive control. The cell lysate was prepared and Western blotted with the phospho-specific anti-Akt (4E2) antibody (first panel), an antitotal Akt antibody (second panel), an anti-PARP antibody (third panel), and an anti-actin antibody (fourth panel). All the experiments were repeated at least three times, and representative blots are shown. tion of NF-B strongly protects cells from both TNF␣-induced apoptosis and apoptosis in response to ionizing radiation and chemotherapeutic agents (44,45). ETOP activates NF-B in HT1080I cells, and such activation modulates apoptosis, apparently through the induction of a Bcl-2-like protein, A1 28 . Consequently, it seems that cells can initiate a counter-reaction for proliferation by utilizing apoptosis and vice versa when appropriate signal pathways are activated. Balancing proliferation and apoptosis might be a fail-safe mechanism for cells to prevent either excessive proliferation or apoptosis. Therefore, an outcome of survival or death may depend on signal strength and context in response to an apoptotic stimulus.
Besides NF-B, another major survival factor impacting the apoptotic core machinery is protein kinase B/Akt. In contrast to NF-B, the activation of Akt in response to apoptotic stimuli has not been reported. This study examines the role of the Akt/protein kinase B pathway in cellular apoptosis induced by two diverse mitochondrial apoptotic stimuli: the potent protein kinase inhibitor (STS) and the DNA damage reagent (ETOP). These agents were chosen because overexpression of Akt has been demonstrated to impair apoptosis induced by either STS (46) or ETOP (10). In response to STS and ETOP, Akt was activated. Although Akt activation occurs early after stimulation, 8 h for STS and 2 h for ETOP, large scale apoptosis begins after 8 h. This is consistent with the theme that the apoptotic machinery has to overcome survival functions to execute cells and is supported by and consistent with reports of late Akt cleavage in certain apoptotic settings (3). More importantly, we have demonstrated that this activation has a functional importance in that it delays and diminishes PARP cleavage and apoptosis (Figs. 3 and 4).
The PI3K/Akt pathway promotes cell survival through a number of mechanisms. Antiapoptotic signals mediated through this pathway include the inactivation of Bad (8) and caspase-9 (9) and the stimulation of NF-B activity (10 -12). Akt-mediated inhibi- . Akt activation was determined by Western blot using the phospho-specific anti-Akt antibody. The PARP and actin levels in these cells were also examined by Western blot using specific antibodies (A). con, control.
FIG. 6. Overexpression of Bcl-2/Bcl-x L does not inhibit STSinduced Akt activation. NIH 3T3 cells were infected with retrovirus expressing either LacZ, human Bcl-2, or human Bcl-x L . The expression of Bcl-2 or Bcl-x L was determined by Western blot using specific antibodies (A). These cells were then either mock-treated or treated with 1 M STS for 8 h. Akt activation was examined by Western blot using the phospho-specific anti-Akt antibody (B). The actin level in these cells is also shown (B). tion of apoptosis seems to occur at a postmitochondrial level, given that Akt overexpression can prevent apoptosis initiated by the microinjection of cytochrome c (47,48), although others have found that Akt prevents the release of cytochrome c from mitochondria (34). We find that Akt activation by STS occurs upstream of mitochondria, i.e. outside of apoptotic core machinery, in that it is not prevented by either caspase inhibition (Fig. 5A) or Bcl-2/Bcl-x L overexpression (Fig. 6B). Consequently, we would not expect that all mitochondrial apoptotic signals activate Akt. This is consistent with our observation that no Akt activation is detected in NIH 3T3 cells treated with either UV or ionizing radiation (data not shown) and reflects observations of NF-B activation in the death receptor apoptotic pathway. Except for the clear protective role of NF-B activation in TNF␣ induced apoptosis, its role in other death receptor-induced apoptosis is not clear.
These data have implications for cellular transformation. A large and growing number of human tumors are associated with PI3K/Akt amplification or activation including cervical (49), ovarian (16), breast (17,18), and prostate cancers (19), and Akt activity often correlates with more malignant behavior (18). The data presented here add one more potential indication of the importance of Akt activity in tumor progression. Therapy-resistant cancers arising after radiotherapy or chemotherapy present major problems in cancer therapy. Chemotherapeutic agents like ETOP target the mitochondrial apoptotic pathway to execute tumor cells. The ability of ETOP to also activate Akt might lead to the emergence of cancers resistant to therapy. In this regard, overexpression of Akt in ovarian cancer cells led to paclitaxel resistance, which is associated with Bad inactivation and a failure of apoptosis (22), and the overexpression in lung cancer cells resulted in topotecan resistance (21). Conversely, the inhibition of PI3K/Akt signaling with wortmannin enhanced pancreatic carcinoma cell sensitivity to gemcitabine (50). If chemotherapy agents such as ETOP induce Akt activity, this is likely to inhibit their desired antitumor effects, particularly in lines in which PI3K/Akt is amplified. The inhibition of Akt activity in these situations might help overcome drug resistance.
In view of this, it is important to understand precisely how Akt is activated. Presently, this is not totally clear. Activation requires recruitment to the plasma membrane by ligation of the N-terminal PH-domain to the PI3-kinase products PI 3,4-P 2 and inositol 1,4,5-trisphosphate. This brings Akt in close proximity to 3-phosphoinositide-dependent protein kinase (PDK)-1, resulting in the phosphorylation of Thr-308 in the T loop of Akt. Phosphorylation of the Ser-473 in the C-terminal hydrophobic motif is also required for Akt activation, but the putative kinase PDK2 that phosphorylates Ser-473 of Akt has not been clearly identified (30). The debate whether PDK1 could also phosphorylate Ser-473 of Akt was settled in the negative by the demonstration of inhibition of phosphorylation of Thr-308 but not Ser-473 in PDK1 Ϫ/Ϫ mouse embryonic stem cells in response to insulin-like growth factor-1 (51). Regardless of the identity of PDK2, our data indicate that STS at 1 M does not inhibit its activity. Because STS inhibits numerous kinases with IC 50 values within the nM or sub-nM range, this finding might offer valuable information for searching this important PDK2.
The data presented here indicate that apoptosis-inducing agents including the chemotherapeutic drug etoposide also independently activate PI3K/Akt, and such activation delays apoptosis. Further work will be required to determine the precise mechanism of activation of PI3K/AKt by these agents and the effect of preventing it.