Agent and cell-type specificity in the induction of insulin resistance by HIV protease inhibitors
Ben-Romano, Ronita; Rudich, Assafd; Török, Dórad; Vanounou, Sharona; Riesenberg, Klarisb; Schlaeffer, Franciscb; Klip, Amirad; Bashan, Navaa,c
From the aDepartment of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva the bInfectious Disease Unit, and the cChildren Metabolic Laboratory, Soroka Medical Center, Beer-Sheva, Israel and the dProgramme in Cell Biology, the Hospital for Sick Children, Toronto, Ontario, Canada.
Requests for reprints to: Dr N. Bashan, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, IL-84105, Israel. Email: firstname.lastname@example.org
Received: 28 March 2002; revised: 8 July 2002; accepted: 28 August 2002.
Objective: To test agent and cell-type specificity in insulin resistance induced by prolonged exposure to HIV protease inhibitors (HPI), and to assess its relation to the direct, short-term inhibition of insulin-stimulated glucose uptake.
Methods: Following prolonged (18 h) and short (5–10 min) exposure to HPI, insulin-stimulated glucose transport, protein kinase B (PKB) phosphorylation, and GLUT4 translocation were evaluated in 3T3-L1 adipocytes, fibroblasts, L6 myotubes, and L6 cells overexpressing a myc tag on the first exofacial loop of GLUT4 or GLUT1.
Results: Prolonged exposure of 3T3-L1 adipocytes to nelfinavir, but not to indinavir or saquinavir, resulted in increased basal lipolysis but decreased insulin-stimulated glucose transport and PKB phosphorylation. In addition, impaired insulin-stimulated glucose uptake and PKB phosphorylation were also observed in the skeletal muscle cell line L6, and in 3T3-L1 fibroblasts. Interestingly, this coincided with increased basal glucose uptake as well as with elevated total-membrane glucose transporter GLUT1 protein content. In contrast to these unique effects of nelfinavir, the mere presence of any of the agents in the 5 min transport assay inhibited insulin-stimulated glucose-uptake activity. This appeared to be caused by direct and specific interaction of the drugs with GLUT4 fully assembled at the plasma membrane, since insulin-stimulated cell-surface exposure of an exofacial myc epitope on GLUT4 was normal.
Conclusions: Independent mechanisms for HPI-induced insulin resistance exist: prolonged exposure to nelfinavir interferes with insulin signaling and alters cellular metabolism of adipocytes and muscle cells, whereas a direct inhibitory effect on insulin-stimulated glucose uptake may occurs through specific interaction of HPI with GLUT4.
Highly active antiretroviral therapy (HAART) regimens containing HIV-1 protease inhibitors (HPI) have a significant beneficial impact on HIV plasma viremia, immune function, opportunistic infections and mortality from AIDS [1,2]. However, a growing body of evidence suggests that up to 60% of treated patients develop a complex of metabolic alterations, to varying degrees, which is emerging as a significant medical concern [3–5]. These alterations include fat tissue redistribution (peripheral lipodystrophy and increased central adiposity), dyslipidemia and systemic insulin resistance [6–11]. The last is characterized clinically by elevated fasting insulin and/or C-peptide levels [3,9,10], and was confirmed using euglycemic–hyperinsulinemic clamp studies . The clinical significance of these side-effects is the exposure of patients to an apparent increased risk for developing premature cardiovascular morbidity, as well as to new-onset or aggravated type 2 diabetes [10,13,14]. Although metabolic alterations have also been described in HPI-sparing HAART regimens [15–19], an increasing amount of data attributes a role for this class of drugs in the induction of insulin resistance [6,9,10,20–22]. HIV-positive subjects receiving HPI became insulin resistant even in the absence of changes in body composition . A role for the HIV infection itself could be ruled out as the cause for this adverse metabolic effect, since healthy (seronegative) individuals treated for 4 weeks with indinavir also developed insulin resistance . Moreover, even a single dose of indinavir to either normal rats  or healthy volunteers  acutely decreased insulin-stimulated glucose disposal during a euglycemic–hyperinsulinemic clamp. However, although the metabolic side-effects of HPI are largely considered to be a class effect, it is not clear whether the various agents have different tendencies to cause metabolic alterations. Furthermore, the pathophysiological and cellular mechanisms responsible for these effects are not clear.
While adipose tissue is likely a key player in the metabolic syndrome induced by HPI, clinical data suggest decreased insulin responsiveness of both liver and skeletal muscle . Clinical, in vivo studies cannot conclusively determine whether the various tissues directly and independently become insulin resistant in response to HPI or whether they are affected secondarily to other changes. At the cellular level, studies utilizing different cell types, agents, and incubation protocols suggested three separate putative mechanisms to explain the induction of insulin resistance by HPI: (i) inhibition of pre-adipocyte to adipocyte differentiation [27–31] and induction of mature adipocyte apoptosis , (ii) direct inhibition of the activity of glucose transporters (GLUT1 and GLUT4) at the plasma membrane , and (iii) impaired insulin signaling [33,34]. Exposure of 3T3-L1 adipocytes to nelfinavir has resulted in increased basal lipolysis and alterations in the regulation of glucose uptake into the cells . Both elevated basal glucose transport activity and impaired response to acute insulin stimulation could be observed. This could be explained by impaired insulin-induced translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane, potentially caused by defective activation of protein kinase B (PKB/Akt) .
The present study was conducted to test agent and cell-type specificity in the induction of insulin resistance by prolonged exposure to HPI and to assess the relation of this effect to the direct inhibition of insulin-stimulated glucose uptake previously reported with short-term exposure to indinavir [26,32,35]. Three HPI were studied: nelfinavir, which has been shown to activate lipolysis and inhibit insulin signaling toward GLUT4 translocation , indinavir, which was suggested to inhibit GLUT4 activity , and saquinavir (commonly used in HPI-containing HAART regimens), which has been shown to inhibit lipoprotein lipase activity, lipid synthesis and insulin-stimulated glucose transport .
Several cell types were used in this study. 3T3-L1 pre-adipocytes (fibroblasts) were obtained from American Tissue Culture Collection and were grown and differentiated to adipocytes exactly as previously described [37,38]. Parental L6 myotubes, as well as L6 cell lines stably expressing either a myc-tagged GLUT4 (L6-GLUT4myc), or a myc-tagged GLUT1 (L6-GLUT1myc), were grown as described previously [39–41].
Treatments with HIV protease inhibitors
HPI were prepared in stock solutions. Nelfinavir (Roche Pharmaceuticals, Tel Aviv, Israel) was prepared at 100 mmol/l concentration in 100% ethanol, saquinavir (Roche Pharmaceuticals) was prepared as a 100 mmol/l solution in dimethyl sulfoxide, and indinavir (Merck Sharp and Dome, Petach Tikva, Israel) was dissolved in water (100 mmol/l). Maximal final ethanol or DMSO concentrations were 0.05%, neither of which had any effect on the various parameters measured in the comparisons of treated and untreated cells. Two treatment protocols with HPI were used. Prolonged treatment was performed for 18 h in serum-free medium supplemented with 0.5% radioimmunoassay grade bovine serum albumin (BSA), or in the case of L6-GLUT4myc myotubes, 15 h with medium supplemented with 10% fetal calf serum followed by an additional 3 h in BSA-supplemented medium. Then, medium from 3T3-L1 adipocytes was collected for measurements of free fatty acids (FFA) and glycerol, and cells were then rinsed with phosphate-buffered saline (PBS) and stimulated with 100 nmol/l insulin in the absence of HPI. Short-term treatment consisted of serum starving the cells for 18 h in the presence of BSA and stimulated for 20 min with 100 nmol/l insulin, after which HPI was added only to the glucose transport solution, or for an equivalent duration (5 min) for translocation experiments. Cell viability was assessed by protein recovery (Bio-Rad, Hercules, California, USA), as well as by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test.
Hexose transport measurements
2-Deoxyglucose (2DG) uptake was performed after rinsing the cells with PBS and incubating them with or without 100 nmol/l insulin for 20 min, exactly as previously described , using 50 and 10 μmol/l 2-deoxy-(3H)-glucose (3.7 × 104 Bq/ml; Nuclear Research Center, Dimona, Israel) for adipocyte and myotubes, respectively.
For basal lipolysis measurements, glycerol and FFA were measured in the medium. FFA concentrations were determined using a commercial kit (Roche, Mannheim, Germany), following the instructions of the manufacturer, and calculated according to a palmitic acid standard curve. Glycerol was measured spectrophotometrically using the GPO trinder kit (Sigma, St Louis, Missouri, USA).
GLUT4myc translocation assay
The amount of surface GLUT4myc was measured in intact (non-permeabilized) L6-GLUT4myc myotubes using an antibody-coupled colorimetric assay as described previously . Briefly, after 20 min incubation with or without 100 nmol/l insulin, cells were washed three times with ice-cold PBS, blocked with 5% goat serum (20 min, 4°C), and anti-myc monoclonal antibody (1:100 dilution; 9E10; Santa Cruz Biotechnology, Santa-Cruz, California, USA) was added for 60 min at 4°C. Cells were then fixed (4% paraformaldehyde for 10 min on ice), and peroxidase-conjugated donkey anti-mouse IgG (1:1000 dilution; Santa Cruz Biotechnology) was added for 60 min at 4°C. After extensive washing, o-phenylenediamine dihydrochloride reagent (Sigma) was added for 30 min at room temperature, and the reaction was stopped by adding 3 mol/l HCl. The supernatant was then collected and absorbance was measured at 492 nm. Background absorbance obtained in the absence of anti-myc antibody was subtracted.
Cell lysates, total membranes and Western blot analysis
Cells (1–2 wells of a 6-well plate per condition) were rinsed three times with PBS. Total membranes were prepared following homogenization of the cells in 250 mmol/l sucrose, 20 mmol/l HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) pH 7.4, 1 mmol/l ethylenediaminetetraacetic acid (EDTA), 0.2 mmol/l sodium vanadate, and inhibitors [1:1000 dilution of protease inhibitor cocktail (Sigma)] and analyzed for GLUT1 content by Western blot analysis using anti-GLUT1 antibody (Chemicon, Temecula, California, USA) as previously described . For Western blot analyses of PKB and pPKB, cell lysates were prepared in 50 mmol/l TRIS HCl pH 7.5, 0.1% (w/v) Triton X-100, 1 mmol/l EDTA, 1 mmol/l ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′- tetraacetic acid, 50 mmol/l sodium fluoride, 10 mmol/l sodium β-glycerophosphate, 5 mmol/l sodium pyrophosphate, 1 mmol/l sodium vanadate, 0.1% (v/v) 2-mercaptoethanol, supplemented with inhibitors as above. After centrifugation of the lysates (12 000 × g, 15 min 4°C), protein concentration was determined in the supernatant using Bio-Rad reagent and Western blot analyses were performed using anti-PKB (PH domain; Upstate Biotechnology, Lake Placid, USA) or anti-phosphoserine-473 PKB (Cell Signaling, Beverly, Massachusetts, USA), as previously described .
Student's t-test was used for comparing values of two groups, and ANOVA followed by post-hoc analysis was used for comparing multiple values. A P value of 0.05 was considered the limit for statistical significance.
Agent specificity in inducing insulin resistance and increased basal lipolysis in 3T3-L1 adipocytes
Prolonged incubation (18 h) of 3T3-L1 adipocytes with 30 μmol/l of a HPI (nelfinavir, indinavir and saquinavir) was followed by insulin stimulation and assay for 2DG uptake in the absence of the drugs. As previously described , fully differentiated 3T3-L1 adipocytes treated with nelfinavir using this protocol exhibited increased basal and decreased insulin-stimulated glucose uptake (Fig. 1a). Interestingly, equimolar concentrations of indinavir or saquinavir failed either to induce enhanced basal glucose-uptake activity or to impair significantly the response to insulin. Likewise, while 18 h nelfinavir treatment resulted in a significant increase in glycerol and FFA release to the culture medium, neither effect was noted with indinavir or saquinavir at 30 μmol/l concentration (Fig. 1b). The effect of the various HPI on insulin-stimulated PKB phosphorylation (a key enzyme in insulin signaling towards GLUT4 translocation) was assessed (Fig. 1c). In quantification analyses, insulin-induced Ser-473 phosphorylation was reduced in nelfinavir-treated cells by 80 ± 4% compared with control cells, whereas the total cellular PKB content was unaltered. Consistent with the glucose transport data presented in Fig. 1a, indinavir and saquinavir at the same concentration failed to induce a decrease in insulin-stimulated PKB phosphorylation. When saquinavir concentration was increased to 50 μmol/l, insulin-stimulated PKB phosphorylation tended to be decreased (data not shown). Collectively, these data demonstrate the potency of prolonged exposure to nelfinavir to induce alterations in glucose uptake, impair insulin response and increase basal lipolysis, compared with the effects of indinavir and saquinavir.
Previous studies have demonstrated the capacity of various HPI to inhibit 2DG uptake in 3T3-L1 adipocytes when present after insulin stimulation and only during glucose-uptake measurements, an effect that was not associated with impaired insulin-stimulated PKB phosphorylation . These data suggested a direct effect of HPI on glucose transporters present at the plasma membrane. Figure 2c demonstrates that the three HPI tested were indeed capable of interfering with insulin-stimulated glucose uptake using this protocol in 3T3-L1 adipocytes (Fig. 2) as well as in L6 muscle cells (Fig. 2b), two cell lines expressing insulin-responsive glucose transporter GLUT4. To assess whether this effect of HPI is dependent on the expression of this isoform, L6 cells were used that overexpressed either GLUT4 or GLUT1, each tagged at their first exofacial loop with a myc epitope [40,41]. In GLUT4myc L6 cells, both basal and insulin-stimulated glucose uptake depend largely on GLUT4, which is expressed at approximately 100-fold over the endogenous GLUT4 or GLUT1 levels. In contrast, GLUT1myc myoblasts are deficient in GLUT4 since the endogenous gene is only expressed upon myoblast to myotube differentiation. Figure 3a demonstrates that in L6-GLUT4myc myotubes indinavir had a marked inhibitory effect on both basal and insulin-stimulated glucose-uptake activity when present only after insulin stimulation in the transport solution. In contrast, L6-GLUT1myc myoblasts were nearly completely resistant to the acute inhibitory effect of nelfinavir or indinavir on glucose uptake (non-significant versus control cells; Fig. 3b). Interestingly, indinavir was more potent than nelfinavir in this acute inhibition of glucose-uptake activity, (Figs 2a and 3a), in contrast to the effects observed with prolonged exposure to HPI (Fig. 1). To verify further that the acute effect of HPI mechanistically differs from the insulin resistance induced by prolonged nelfinavir, insulin-stimulated GLUT4myc translocation was assessed. L6-GLUT4myc myotubes were stimulated by insulin, incubated with HPI for 5 min, after which the external exposure of the myc epitope in non-permeabilized cells was measured. As shown in Fig. 3c, neither indinavir nor nelfinavir, had any effect on the translocation and full insertion of GLUT4 in the plasma membrane following insulin stimulation, despite reducing glucose uptake. This is in contrast to the effect of prolonged nelfinavir treatment of these cells, which resulted in 80% inhibition of insulin-stimulated GLUT4myc translocation (not shown), consistent with previous results in 3T3-L1 adipocytes . Collectively, these data suggest that, while various HPI share the capacity to inhibit glucose uptake acutely by interfering with GLUT4 activity, only nelfinavir at the concentrations tested is capable of inducing metabolic alterations and impairing insulin action following prolonged treatment.
Cell-type specificity in the induction of insulin resistance by nelfinavir
To gain insight on the cell-type specificity of prolonged nelfinavir exposure, 3T3-L1 pre-adipocytes (fibroblasts), as well as L6 myotubes were used. The MTT mitochondrial dye-based assay was used to assess the sensitivity of the different cell types to the cytotoxic effects of 18 h exposure to nelfinavir. While cell viability of differentiated 3T3-L1 adipocytes was unaffected by 40 μmol/l nelfinavir or less, 3T3-L1 pre-adipocytes were resistant (even 100 μmol/l nelfinavir was not associated with significant cell loss) and L6 myotubes were more sensitive to the cytotoxic effect of nelfinavir (significant viability loss was observed with 30 μmol/l nelfinavir). Based on these findings, the non-cytotoxic range of the drug for each cell type was used for further analysis. In all cell types, a dramatic increase in basal glucose uptake was observed following 18 h exposure to nelfinavir, associated with a decrease in the capacity of insulin to increase 2DG uptake activity further (Fig. 4a). Since GLUT1 is the only glucose transporter expressed in 3T3-L1 pre-adipocytes , and GLUT4 was unaltered in 3T3-L1 adipocytes by 18 h treatment with nelfinavir , the increase in basal glucose-uptake activity was examined for an association with elevated GLUT1 levels in the three cell types tested. Figure 4b demonstrates that total membrane GLUT1 protein content was increased by 2.5 ± 0.2, 3.5 ± 0.6 and 1.9 ± 0.3 fold in 3T3-L1 adipocytes, pre-adipocytes and L6 myotubes, respectively. These results suggest that increased protein content of GLUT1 may be involved in the increase in basal glucose uptake induced by 18 h nelfinavir treatment in various cell types.
The Ser-473 phosphorylation of PKB by insulin was used as a marker for the induction of insulin resistance by 18 h nelfinavir in the various cell types. As shown in Fig. 5, non-cytotoxic nelfinavir concentrations impaired insulin-stimulated PKB phosphorylation in 3T3-L1 pre-adipocytes and in L6 myotubes, as shown previously for 3T3-L1 adipocytes (Fig. 1, ), without altering its phosphorylation state in the absence of insulin. Yet, the cell types differed in both the degree of impairment in PKB phosphorylation, which was tolerated without cytotoxicity, and in the median effective dose for the maximal effect observed in each cell type. Maximal degree of inhibition of insulin-stimulated PKB phosphorylation was > 80 ± 5% in 3T3-L1 adipocytes and pre-adipocytes, but only 60 ± 9% in L6 myotubes.
Adipose tissue is a key target for HPI in inducing insulin resistance, as the metabolic syndrome described in patients taking HAART consists of gross alterations in fat tissue distribution and dyslipidemia, including elevated circulating FFA and glycerol levels . Adipose tissue is now believed to play a major role in the pathogenesis of systemic insulin resistance. In particular, FFA and various adipose-derived peptides (adipokines) have been identified as modulators of whole-body insulin sensitivity [43–46]. The systemic insulin resistance seen in patients taking HAART also involves impaired insulin responsiveness in skeletal muscle and liver . Consequently, whether these are secondary to the alterations induced by HPI in adipose tissue or represent direct effect(s) of HPI in different tissues is of major interest.
The present study investigated the agent and cell-type specificity of HPI in inducing insulin resistance, focusing on two potential cellular mechanisms. The data presented demonstrate that, while nelfinavir, indinavir and saquinavir can directly inhibit glucose uptake when present acutely following insulin stimulation in cells expressing GLUT4, they manifest differently when used in a prolonged incubation period. Under the prolonged conditions used, only nelfinavir elevated basal lipolysis, increased basal glucose uptake associated with elevated GLUT1 expression, and impaired insulin-stimulated glucose uptake and PKB phosphorylation. These effects of prolonged exposure to nelfinavir could be observed in differentiated 3T3-L1 adipocytes, 3T3-L1 pre-adipocytes and in L6 myotubes. Therefore, HPI at the same concentrations that cause insulin resistance and metabolic alterations in adipocytes, also affects non-adipocyte cells including skeletal muscle cells. It is, therefore, conceivable that the reported HPI-induced insulin resistance of skeletal muscle and liver  may represent a combination of direct effects of HPI in these tissues and actions secondary to adipose tissue alterations.
At the cellular level, the mechanism for HPI-induced insulin resistance and lipodystrophy appears to be complex. The current literature suggests the existence of at least three mechanisms: (i) direct inhibition of GLUT4 activity [32,47]. (ii) activation of basal lipolysis [34,36,48] and inhibition of insulin signals towards GLUT4 translocation , and (iii) interference with pre-adipocyte to adipocyte differentiation [27–30]. The three mechanisms vary for each agent and time of exposure to the drug. The direct inhibition of GLUT4 activity is common to all HPI tested ( and Figs 2 and 3) and occurs within minutes, even when the agents are present after insulin stimulation and only in the transport solution. This appears to be a direct but reversible interaction between the HPI and the glucose transporters present on the plasma membrane, since this effect is eliminated upon removal of the agents. Our data are consistent with the notion that the GLUT4 isoform is preferentially vulnerable to this acute inhibition by HPI, since this effect was observed only in GLUT4-expressing cells [3T3-L1 adipocytes, wild-type L6 myotubes (Fig. 2) and L6-GLUT4myc (Fig. 3a)]. In contrast, cells lacking GLUT4 and expressing GLUT1myc were resistant to acute inhibition of glucose-uptake activity by HPI (Fig. 3b). Moreover, by assessing GLUT4 translocation through the extracellular exposure of the myc epitope in non-permeabilized cells, it is possible to conclude that the acute effect of HPI does not interfere with the normal assembly and full insertion of the transporters at the plasma membrane. Rather, it is likely to represent a direct interaction between GLUT4 epitopes and the HPI. These results suggest that, in vivo, if the effective tissue HPI concentrations are sufficient, GLUT4-expressing cells like adipocytes , and skeletal muscle cells (Figs 2 and 3 and ) may be vulnerable to this direct effect of HPI.
When exposure to nelfinavir was extended to 18 h incubation, 3T3-L1 adipocytes exhibited increased basal lipolysis and elevated basal but reduced insulin-stimulated glucose uptake . These effects, associated with enhanced total membrane GLUT1 expression (Fig. 4) and impaired capacity of insulin to stimulate PKB phosphorylation (Fig. 5), are likely independent of the direct inhibitory effect that HPI drugs exhibit on glucose uptake, for several reasons. First, the fall in insulin-stimulated glucose uptake following prolonged nelfinavir treatment is observed in the absence of the agent in the transport solution. Second, this lessened insulin action is not restricted to GLUT4-expressing cells, since 3T3-L1 pre-adipocytes and L6-GLUT1myc myoblasts also exhibit comparable effects (Figs 4 and 5). Third, while prolonged nelfinavir impaired insulin-stimulated PKB phosphorylation (Figs 1c and 5) and GLUT4 translocation , the short-term effect of HPI did not interfere with PKB phosphorylation  nor with GLUT4 translocation ( and Fig. 3c).
Since at least the direct inhibition of GLUT4 and the long-term effect of nelfinavir may represent independent cellular mechanisms for insulin resistance, different candidate molecular mediators may be speculated to be involved. While the molecular basis for the direct interaction between HPI and GLUT4 has yet to be identified, several hypotheses may be raised to explain the alterations induced by prolonged exposure to nelfinavir. Increased expression of proteins like SH2-domain-containing 5-phosphatase and/or protein phosphatase 2A, potentially through interference with their normal degradation, could result in altered insulin signaling compatible with the effect of prolonged nelfinavir: impaired PKB activation in the face of normal phosphatidylinositol 3-kinase activation ( and unpublished data). Increased cellular cyclic AMP levels would explain elevated lipolysis , impaired PKB activation by insulin  and increased GLUT1 expression [52,53]. Finally, HPI-induced inhibition of adipocyte differentiation may involve yet other proteins, such as sterol regulatory element binding protein 1 . It is tempting to speculate that HPI drugs inhibit a mammalian protein degradation system resulting in increased expression of proteins, which, in turn, affects lipolysis, insulin action and differentiation. However, so far, such a mechanism has only been shown for the degradation of apolipoprotein B, potentially explaining HPI-induced dyslipidemia .
In conclusion, HPI drugs have the potential to cause insulin resistance and metabolic alterations in various cell types representing targets of insulin action, through diverse and possibly independent mechanisms. Understanding these effects at the cellular and molecular level will be essential for both the design of new HPI with a milder side-effect profile, and the planning of therapeutic strategies to combat the unwanted effects of HAART.
We acknowledge the technical assistance of Evgeniya Malyarevskaya.
Sponsorship: Assaf Rudich is the recipient of the Albert Renold Career Development Award of the European Foundation for the Study of Diabetes. Amira Klip is supported by a Grant-in-Aid, ‘Regulation of the Intrinsic Activity of Glucose Transporters by Insulin', from the Canadian Diabetes Association. Nava Bashan is supported by a grant from the Israel Science Foundation.
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© 2003 Lippincott Williams & Wilkins, Inc.
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