The HIV Protease Inhibitor Nelfinavir Induces Insulin Resistance And

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The HIV Protease Inhibitor Nelﬁnavir Induces Insulin Resistance and Increases Basal Lipolysis in 3T3-L1 Adipocytes Assaf Rudich,1 Sharon Vanounou,2 Klaris Riesenberg,5 Michal Porat,2 Amir Tirosh,2 Ilana Harman-Boehm,3,6 Andrew S. Greenberg,4 Francisc Schlaeffer,5 and Nava Bashan2,3

HIV protease inhibitors (HPIs) are potent antiretroviral agents clinically used in the management of HIV infection. Recently, HPI therapy has been linked to urrent antiretroviral treatment (highly active the development of a metabolic syndrome in which antiretroviral therapy [HAART]) has improved adipocyte insulin resistance appears to play a major the prognosis of patients with HIV by dramati- role. In this study, we assessed the effect of nelfinavir Ccally suppressing HIV viral load, increasing CD4 on glucose uptake and lipolysis in differentiated 3T3-L1 counts, and reducing opportunistic infections associated adipocytes. An 18-h exposure to nelfinavir resulted in with AIDS (1,2). However, it is increasingly recognized an impaired insulin-stimulated glucose uptake and acti- that as much as 83% of the patients receiving this treat- vation of basal lipolysis. Impaired insulin stimulation of ment develop metabolic abnormalities, which include dys- glucose up take occurred at nelfinavir concentrations lipidemia (elevated triglycerides and cholesterol), central ␮mol/l) and could be attributed 20 ؍ ␮mol/l (EC 10 > 50 adiposity, and peripheral lipodystrophy (3,4). Interest- to impaired GLUT4 translocation. Basal glycerol and ingly, treated HIV patients who developed this clustering free fatty acid (FFA) release were significantly en- hanced with as low as 5 ␮mol/l nelfinavir, displaying of metabolic abnormalities were also found to have ele- fivefold stimulation of FFA release at 10 ␮mol/l. Yet, the vated fasting insulin or C-peptide levels (5–7), strongly antilipolytic action of insulin was preserved at this suggesting that these individuals develop systemic insulin concentration. Potential underlying mechanisms for resistance. Moreover, elevated C-peptide levels appear to these metabolic effects included both impaired insulin be a reliable predictor for the development of lipodystro- stimulation of protein kinase B Ser 473 phosphorylation phy. Importantly, it is becoming increasingly recognized with preserved insulin receptor substrate tyrosine that these patients may be at high risk for developing phosphorylation and decreased expression of the lipol- premature cardiovascular morbidity as well as type 2 dia- ysis regulator perilipin. Troglitazone pre- and cotreat- betes, emphasizing the medical significance of the meta- ment with nelfinavir partly protected the cells from the bolic dysregulation associated with HAART (4,7,8). increase in basal lipoysis, but it had no effect on the Which component of the antiretroviral regimen, or impairment in insulin-stimulated glucose uptake in- whether the HIV infection itself, is responsible for this duced by this HPI. This study demonstrates that nelfi- seemingly serious abnormality is not fully clear. AIDS is navir induces insulin resistance and activates basal frequently associated with hypertriglyceridemia, but with lipolysis in differentiated 3T3-L1 adipocytes, providing potential cellular mechanisms that may contribute to reduced total, LDL, and HDL cholesterol (9,10). Nucleo- altered adipocyte metabolism in treated HIV patients. side reverse transcriptase inhibitors may also be associ- Diabetes 50:1425–1431, 2001 ated with hepatic steatosis and lactic acidemia, which are thought to result from mitochondrial damage caused by these agents (11). Yet, an increasing amount of clinical and epidemiological data attributes a central role for the HIV protease inhibitors (HPIs) in the induction of insulin From the 1S. Daniel Abraham Center for Health and Nutrition, the 2Department of resistance in HAART-treated patients (3,5,6,12,13). HPIs Clinical Biochemistry, and the 3Leslie and Susan Gonda (Goldschmied) Labora- tory for Multi-Disciplinary Diabetes Research, Ben-Gurion University of the are a central component of the HAART regimen, as they Negev, Beer-Sheva, Israel; the 4Jean Mayer USDA Human Nutrition Research are potent inhibitors of HIV aspartyl protease, an enzyme Center on Aging at Tufts University, Boston, Massachusetts; the 5Infectious Disease Unit and the 6Diabetes Unit, Soroka Medical Center, Beer-Sheva, Israel. required for normal processing of HIV proteins (14–16). It Address correspondence and reprint requests to Nava Bashan, PhD, De- appears that all HPI compounds induce insulin resistance partment of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion and lipid abnormalities to various degrees (3,17), but the University of the Negev, Beer-Sheva, IL-84105, Israel. E-mail: nava@bgumail. bgu.ac.il. mechanisms for these serious side effects are largely Received for publication 16 February 2001 and accepted 15 March 2001. unknown. A.R. and S.V. contributed equally to this work 2DG, 2-deoxyglucose; BSA, bovine serum albumin; DMEM, Dulbecco’s Only a few studies have been published recently regard- modified Eagle’s medium; FFA, free fatty acid; HAART, highly active anti- ing alterations in insulin signaling and insulin-regulated retroviral therapy; HPI, HIV protease inhibitor; HSL, hormone-sensitive lipase; metabolism caused by HPIs at the cellular level. Using IRS, insulin receptor substrate; KRPB, Krebs-Ringer phosphate buffer; PBS, phosphate-buffered saline; PKB, protein kinase B; RIA, radioimmunoassay; different agents and incubation periods, protease inhibi- TNF, tumor necrosis factor; TZD, thiazolidinedione. tors have been shown to impair insulin signaling events in

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HepG2 cells (18), to inhibit adipocyte differentiation (19, (4G10) antibodies (purchased from Upstate Biotechnology [Lake Placid, NY]); 20), and to decrease adipocyte insulin-stimulated glucose anti–Ser 473 PKB antibody (from Cell Signaling [Beverly, MA]); and anti– hormone-sensitive lipase (HSL) and anti-perilipin antibody recognizing both transport without affecting the insulin signaling cascade the A and B isoforms (prepared as previously described) (22). (21). Yet, despite the fact that impaired adipose tissue Immunofluorescent detection of GLUT4 in plasma membrane lawns. metabolism is at the center of the HPI-induced metabolic Cells cultured on glass coverslips in six-well plates were treated for 18 h syndrome, a systematic evaluation of the effects of HPIs without or with 30 ␮mol/l nelfinavir and subsequently rinsed once in ice-cold PBS, followed by a 30-s incubation on ice with 0.5 mg/ml of poly-L-lysine in on fully differentiated adipocyte metabolism has not been PBS. The cells were then swollen in a hypotonic buffer (23 mmol/l KCl, 10 reported. mmol/l HEPES, pH 7.5, 2 mmol/l MgCl2, and 1 mmol/l EGTA) by three In this study, we investigated the effect of nelfinavir, a successive rinses. Thereafter, cells were sonicated for 5 s using a 5-mm clinically used HPI, on glucose transport and lipolysis in microtip set 1 cm above the surface of the cell monolayer in 10 ml of sonication buffer (70 mmol/l KCl, 30 mmol/l HEPES, pH 7.5, 5 mmol/l MgCl , differentiated 3T3-L1 aidpocytes, with emphasis on insulin 2 3 mmol/l EGTA, 1 mmol/l dithiothreitol, and 0.1 mmol/l phenylmethylsulfonyl responsiveness. We demonstrate that 18 h of exposure to fluoride). The bound plasma membrane sheets were washed two times with nelfinavir results in increased basal lipolysis, an effect as- sonication buffer; fixed for 20 min at 4°C in a solution containing 2% sociated with reduced expression of perilipin, a protein paraformaldehyde, 70 mmol/l KCl, 30 mmol/l HEPES, pH 7.5, 5 mmol/l MgCl2, thought to be important in the regulation of lipolysis (22, and 3 mmol/l EGTA; and incubated for an additional 15 min at 25°C in 100 mmol/l glycine-PBS (pH 7.5). After three rinses with PBS, the sheets were 23). In addition, with higher concentrations, nelfinavir im- blocked overnight at 4°C in PBS containing 5% goat serum (Zymed lab, San pairs insulin stimulation of glucose transport, protein ki- Francisco, CA) and further incubated at 4°C overnight with a 1:500 dilution of nase B (PKB) phosphorylation, and GLUT4 translocation. the GLUT4 antibody. The plasma membrane sheets were then washed six times with PBS and incubated overnight at 4°C with a 1:500 dilution of Cy3-conjugated AffiniPure goat anti–rabbit IgG (Jackson Immunoresearch, RESEARCH DESIGN AND METHODS Baltimore, PA). After incubation with the secondary antibodies, the mem- Cell culture and treatments. 3T3-L1 adipocytes (American Type Culture brane sheets were washed six times with PBS and mounted for microscopic Collection) were grown in Dulbecco’s modified Eagle’s medium (DMEM) analysis with Vectashield mounting medium (Vector Laboratories). Laser (Biological Industries, Beit-Haemek, Israel) and differentiated exactly as confocal microscopy images were obtained using a LSM 510 Zeiss (Ober- previously described (24,25). Fully differentiated cells (12–14 days after kochen, Germany) laser confocal microscope. induction of differentiation) were incubated in serum-free DMEM supplement- Statistical analysis. The Student’s t test was used for comparing values of ed with 0.5% radioimmunoassay (RIA)-grade bovine serum albumin (BSA) two groups. P ϭ 0.05 was considered the limit for statistical significance. (Sigma, St. Louis, MO), with or without various concentrations of nelfinavir, for 18 h. For stock solution, nelfinavir (Roche Pharmaceuticals, Tel Aviv, Israel) was prepared at 100 mmol/l concentration in 100% ethanol, resulting in RESULTS maximal final ethanol concentration of 0.04%. Cell viability, as assessed by Effect of nelfinavir on glucose transport and glucose protein recovery and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro- mide test, was unaffected with up to 40 ␮mol/l nelfinavir. For pre- and co- transporters. Fully differentiated 3T3-L1 adipocytes were treatment with troglitazone (10 ␮mol/l, provided by Drs. Alan Saltiel and Heidi exposed for 18 h to the indicated nelfinavir concentrations Camp, Parke-Davis, Ann-Arbor, MI), cells were incubated in full DMEM for 6 h as described in RESEARCH DESIGN AND METHODS, after which before and then in serum-free medium during nelfinavir treatment. 2DG uptake was measured (Fig. 1A). Although 10 ␮mol/l Hexose transport measurements. 2-Deoxyglucose (2DG) uptake was performed after rinsing the cells with phosphate-buffered saline (PBS) and nelfinavir treatment was not associated with any signifi- incubating them without or with 100 nmol/l insulin for 20 min, exactly as cant change, higher concentrations were associated with previously described (24,26), using 50 ␮mol/l 2-deoxy-[3H]glucose (Nuclear elevated basal levels and gradually reduced insulin-stimu- Research Center, Dimona, Israel) (3.7 ϫ 104 Bq/ml) for 10 min. lated 2DG uptake. With 30 ␮mol/l nelfinavir, net insulin Lipolysis determinations. For basal lipolysis measurements, glycerol and effect was reduced from 310 Ϯ 19 in control cells to 54 Ϯ free fatty acids (FFAs) were measured in the medium after the 18-h incuba- Ͻ tion. For other studies of FFA release, cells were rinsed three times with PBS 11 pmol 2DG/mg protein per min (P 0.001), with an ϳ ␮ (after the 18-h nelfinavir treatment) and further incubated for 1 h with Krebs- EC50 of 20 mol/l. To investigate whether decreased Ringer phosphate buffer (KRPB) (HEPES 50 mmol/l, pH 7.4, NaCl 128 mmol/l, abundance of the insulin-responsive glucose transporter KCl 4.7 mmol/l, CaCl 1.25 mmol/l, MgSO 1.25 mmol/l, and sodium phosphate 2 4 GLUT4 could explain the reduction in insulin-stimulated 10 mmol/l) supplemented with 1% RIA-grade BSA, as previously described (27). For maximally stimulated lipolysis and for insulin anti-lipolysis measure- glucose transport, total membranes were prepared and ments, isoproteranol (Sigma) at either maximal (1 ␮mol/l) or submaximal analyzed by Western blot. Figure 1B demonstrates that to- (5 nmol/l) stimulatory concentrations, respectively, was added during the 1-h tal membrane GLUT4 content was not significantly changed incubation in KRPB in the absence or presence of 100 nmol/l insulin. FFA by nelfinavir at concentrations of 10–40 ␮mol/l. We next concentrations were determined using a commercial kit (Roche, Mannheim, assessed insulin-stimulated GLUT4 translocation by immu- Germany), following the instructions of the manufacturer, and calculated according to a palmitic acid standard curve. Glycerol was measured spectro- nofluorescent detection of GLUT4 in plasma membrane photometrically using a glycerol 3-phosphate oxidase trinder kit (Sigma). lawns. Figure 1C demonstrates that 30 ␮mol/l nelfinavir Cell lysates, total membranes, and Western blots. Cells (one to two wells was not associated with a significant change in plasma of a six-well plate per condition) were rinsed three times with PBS. Total membrane GLUT4 abundance in the non–insulin-stimu- membranes were prepared after homogenization of the cells in 250 mmol/l sucrose, 20 mmol/l HEPES, pH 7.4, 1 mmol/l EDTA, 0.2 mmol/l sodium lated state. Yet, after insulin stimulation, an impairment vanadate, and inhibitors (a 1:1,000 dilution of protease inhibitor cocktail in insulin-stimulated GLUT4 translocation to the plasma [Sigma]), as previously described, and analyzed for GLUT4 content by West- membrane could be observed. ern blot analysis using anti-GLUT4 antibody (Chemicon, Temecula, CA) (24). Nelfinavir alters adipocyte lipolysis. Since HPI treat- For Western blot analyses of other proteins, cell lysates were prepared in 50 ment is associated with both peripheral insulin resistance mmol/l Tris-HCl, pH 7.5, 0.1% (wt/vol) triton X-100, 1 mmol/l EDTA, 1 mmol/l EGTA, 50 mmol/l NaF, 10 mmol/l Na ␤-glycerophosphate, 5 mmol/l sodium as well as peripheral lipodystrophy, which suggests alter- pyrophosphate, 1 mmol/l sodium vanadate, and 0.1% (vol/vol) 2-mercaptoetha- ations in adipocyte lipid metabolism, we next assessed the nol, supplemented with inhibitors as above. After centrifugation of the lysates effects of nelfinavir on adipocyte lipolysis. An 18-h expo- (12,000g for 15 min at 4°C), the fraction between the pellet and the adipose sure to nelfinavir resulted in a marked stimulation of basal cake was collected, and protein concentration was determined using Bio-Rad reagent. Then, 15–50 ␮g protein was resolved on 7.5 or 10% SDS-PAGE and lipolysis, as assessed by the increase of both FFAs and subjected to Western blot analyses using the following antibodies: anti–insulin glycerol levels in the culture medium (Fig. 2A). Interest- receptor substrate (IRS)-1, anti-PKB PH domain, and anti-phosphotyrosine ingly, this effect of nelfinavir could be observed at concen-

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FIG. 1. Nelfinavir impairs insulin-stimulated glucose uptake and GLUT4 translocation. 3T3-L1 adipocytes were incubated for 18 h with the indicated concentrations of the HPI nelfinavir. A: Cells were rinsed and further incubated without or with 100 nmol/l insulin for 20 min, after which 2DG uptake activity was determined. Results are means ؎ SE of five independent experiments performed at least in duplicate. *P < 0.05 vs. untreated cells; †P < 0.05 vs. insulin-stimulated control cells; ‡P < 0.01 vs. insulin-stimulated control cells. B: Western blot analysis of GLUT4 in total membranes of 3T3-L1 adipocytes. The blot shown is representative of two independent experiments yielding the same result. C: Immunofluorescent detection of GLUT4 in plasma membrane lawns prepared from cells treated without (control) or with 30 ␮mol/l nelfinavir, and subsequently stimulated without or with 100 nmol/l insulin for 20 min. Shown is a picture representing similar results obtained in three independent experiments. trations as low as 5 ␮mol/l, whereas 10 ␮mol/l, which did believed to regulate lipolytic activity by limiting the access not alter glucose transport activity (Fig. 1A), was already of HSL to the lipid droplets (28–30). Figure 3A demon- associated with a 5.74 Ϯ 0.70–fold increase in FFA release. strates the effect of nelfinavir on the content of these two To assess whether the insulin resistance observed (Fig. proteins. As shown in the upper blot, HSL protein content 1A) could be similarly demonstrated on the acute anti- was not elevated in nelfinavir-treated cells compared with lipolytic action of insulin, cells were submaximally stim- control cells, and it even tended to decrease with concen- ulated with 5 nmol/l isoproteranol, without or with 100 trations of 30 ␮mol/l. This finding suggests that the in- nmol/l insulin, for 1 h after nelfinavir treatment. As depict- creased basal lipolysis shown above (Fig. 2A) cannot be ed in Fig. 2B, FFA release in control cells was stimulated attributed to increased HSL expression. The lower blot ϳ18-fold by 5 nmol/l isoproteranol and was decreased by demonstrates the protein content of the two isoforms of 58% in the presence of insulin. In cells treated for 18 h with perilipin, A and B (molecular weight 57 and 46 kDa, re- 5 and 10 ␮mol/l nelfinavir, insulin demonstrated a pre- spectively). A gradual decrease in adipocyte perilipin served antilipolytic effect, resulting in 63 and 54% inhibi- content with increasing nelfinavir concentrations could be tion of isoproteranol-stimulated FFA release, respectively. seen, beginning with as low as 10 ␮mol/l nelfinavir, and At 30 ␮mol/l nelfinavir, no significant stimulation of FFA involved both isoforms. release could be demonstrated with 5 nmol/l isoprotera- To investigate the potential mechanisms by which nelfi- nol, and only a nonsignificant 9% decrease could be navir treatment impairs the insulin signaling cascade, cells induced by insulin. Interestingly, in both nelfinavir-treated were incubated with nelfinavir for 18 h and then stimu- and untreated cells, exposure to 1 ␮mol/l isoproteranol lated with 100 nmol/l insulin for 7 min. Tyrosine phosphor- resulted in a further increase in lipolysis rate compared ylation of a band of ϳ185 kDa, corresponding to IRS-1 and with 5 nmol/l isoproteranol, reaching similar rates of FFA –2, is shown in Fig. 3B along with an IRS-1 immunoblot. As release the medium (data not shown). shown, nelfinavir treatment had no effect on either IRS-1 Potential cellular mechanisms for nelfinavir-stimu- content nor on its insulin-stimulated tyrosine phosphory- lated lipolysis and insulin resistance. Long-term regulation. Similarly, insulin-stimulated tyrosine phosphoryla- lation of lipolysis may involve alterations both in the tion of the ␤-subunit of the insulin receptor (ϳ95 kDa expression level of HSL and in perilipins. The latter are band) was unaffected by nelfinavir treatment (not shown),

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Insulin and thiazolidinediones significantly inhibit nelfinavir-stimulated lipolysis. We next assessed whether the induction of basal adipocyte lipolysis by nelfinavir treatment could be blocked by acute insulin treatment or by pre- and coincubation with the thiazolidinedione (TZD) insulin-sensitizer troglitazone. Figure 4A demonstrates that both agents display a significant capacity to inhibit nelfinavir-stimulated FFA release, particularly at lower concentrations of this HPI. At a nelfinavir concentration of

FIG. 2. The effect of nelfinavir treatment on basal lipolysis and the antilipolytic effect of insulin. A: Glycerol and FFA concentrations were determined in the medium of 3T3-L1 adipocytes treated with nelfinavir for 18 h, as described in RESEARCH DESIGN AND METHODS. Results are means ؎ SE of four independent experiments performed at least in duplicate. *P < 0.05 vs. glycerol release in untreated cells; †P < 0.05 vs. FFA release in control cells; ‡P < 0.002 vs. control cells. B: After treatment with nelfinavir for 18 h, cells were rinsed and incubated in KRPB for an additional 1 h without (basal) or with either 5 nmol/l isoproteranol alone or with 100 nmol/l insulin, after which FFA release was assayed. Shown are results of three independent experiments performed at least in duplicate. suggesting that nelfinavir did not impair early steps in the FIG. 3. The effect of nelfinavir on protein content of HSL and perilipin and on IRS-1 and PKB content and insulin-stimulated phosphorylation. insulin signaling cascade. In contrast, insulin-stimulated A: Western blot analyses of HSL and perilipin protein content in total-cell Ser 473 phosphorylation of PKB was significantly inhibited lysates of 3T3-L1 adipocytes treated for 18 h with the indicated nelfi- by nelfinavir concentrations of at least 20 ␮mol/l, without navir concentrations. B: 3T3-L1 adipocytes were treated with nelfinavir as described in A, rinsed, and stimulated for 7 min with 100 nmol/l alterations in its total protein content (Fig. 3C). Densito- insulin, after which cell lysates were prepared. Western blot analyses metric analyses show a dose-dependent effect, which par- using anti–IRS-1 and anti-phosphotyrosine antibodies are presented. C: Cells were treated as described in B, after which Western blot anal- allels the effect observed on 2DG uptake activity (Fig. 1A). ysis for total PKB or for Ser 473 PKB was performed. Results of den- These data suggest a potential role for reduced perilipin sitometry analysis of three independent experiments performed in content in nelfinavir-stimulated lipolysis and an impair- duplicate are shown, with a value of 1 given to the intensity of the band achieved in insulin-stimulated control cells. *P < 0.05 vs. control; †P < ment in insulin-stimulated PKB phosphorylation in the 0.002 vs. control. All shown blots represent at least three independent induction of insulin resistance. experiments yielding similar results.

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take was measured. As shown in Fig. 4B, insulin-stimulated 2DG uptake activity was increased by nearly 20% in troglitazone-treated control cells. This effect of troglitazone was only marginally significant when cells were co- treated with 10 ␮mol/l nelfinavir and was completely absent at 30 ␮mol/l. These results indicate that although troglitazone showed a certain capacity to protect against the metabolic effects of nelfinavir, troglitazone became less efficient with increasing doses of this agent.

DISCUSSION HPI treatment, which dramatically improved the prognosis of HIV-infected patients, appears to cause or aggravate a metabolic syndrome with potentially severe consequences (7). The cellular mechanisms underlying the appearance of this syndrome are largely unknown. The clinical evidence of systemic peripheral insulin resistance, as well as the high prevalence of peripheral lipodystrophy and central adiposity, suggests that altered adipose tissue metabolism may have a central role in the development of this syndrome (4,8). Here, we demonstrate that the HPI nelfinavir, when incubated with 3T3-L1 adipocytes for an 18-h period, results in both increased basal lipolysis as well as resistance to the acute metabolic actions of insulin. These two effects display distinct sensitivities to nelfinavir, with the activation of lipolysis occurring at lower concentrations of nelfinavir than those at which the induction of insulin resistance occurs. A recent study on the effect of HPI on glucose transport activity in differentiated 3T3-L1 adipocytes also reached the conclusion that HPIs impair insulin-stimulated glucose uptake activity (21). This impairment was associated with normal GLUT4 expression as well as with preserved insulin- stimulated tyrosine phosphorylation events. However, although impaired insulin-stimulated GLUT4 translocation associated with decreased PKB Ser 473 phosphorylation was observed in our study (Figs. 1C and 3C), these were reported as normal in the study by Murata et al. (21). This discrepancy may be attributed to the different HPI agents used in the two studies and possibly also to the concentrations and duration of incubation used. Significant activation of basal lipolysis occurred at nelfinavir concentrations as low as 5 ␮mol/l, whereas the HPI effect on insulin-stimulated glucose uptake was only observed at higher concentrations (Fig. 2A). These findings suggest that these two metabolic changes are the result of independent mechanisms. In addition, although it is tempt- FIG. 4. The effect of troglitazone and insulin on basal FFA release and on insulin-stimulated 2DG uptake. A: Cells were either treated for 18 h ing to speculate that the insulin resistance is secondary to with nelfinavir or treated for 5 h before and during nelfinavir treat- the activated lipolysis, the studies with troglitazone do not ment with 10 ␮mol/l troglitazone. Cells were then washed and incu- necessarily support this notion. Although troglitazone at bated for an additional1hinKRPB, either with no additions or with 100 nmol/l insulin. FFA release was then assessed. Results are derived least partly inhibited nelfinavir-induced lipolysis, it did not from three independent experiments performed at least in duplicate. protect against the decrease in insulin-stimulated glucose *P < 0.05 and †P < 0.01 vs. cells treated with the same nelfinavir concentration but without either troglitazone or insulin. B: Cells were uptake. The decreased efficiency of troglitazone with in- treated with nelfinavir without or with cotreatment with troglitazone, creasing nelfinavir concentrations may be consistent with as described in A, rinsed, and stimulated for 20 min with 100 nmol/l a recent report demonstrating decreased peroxisome pro- insulin, after which 2DG uptake was measured. Results are derived ␥ from two independent experiments yielding identical results. liferator–activated receptor- protein content in nelfinavir- treated 3T3-L1 adipocytes (20). This finding may explain the reduced expression of various adipocyte-specific pro- 30 ␮mol/l, only troglitazone provided a mild but significant teins in response to nelfinavir treatment, as well as the inhibition in FFA release. To determine whether troglita- inhibition of the preadipocyte to the adipocyte differenti- zone could also protect against the impairment in insulin ation program (20). In the present study, adipocyte differ- action induced by nelfinavir, insulin-stimulated 2DG up- entiation was not directly assessed. Yet, GLUT4 protein

DIABETES, VOL. 50, JUNE 2001 1429 NELFINAVIR ALTERS ADIPOCYTE METABOLISM content was unaffected by nelfinavir treatment (Fig. 1B), REFERENCES whereas HSL content at the higher range of nelfinavir 1. Shafer RW, Vuitton DA: Highly active antiretroviral therapy (HAART) for used, and perilipin also at lower concentrations, were the treatment of infection with human immunodeficiency virus type 1. Biomed Pharmacother 53:73–86, 1999 found to be reduced (Fig. 3A). 2. Kaplan JE, Hanson D, Dworkin MS, Frederick T, Bertolli J, Lindegren ML, Perilipins, located under basal conditions at the phospho- Holmberg S, Jones JL: Epidemiology of human immunodeficiency virus- lipid interphase of the triglyceride droplet, are believed to associated opportunistic infections in the United States in the era of highly limit HSL access to its substrate (28–30). Consequently, a active antiretroviral therapy. Clin Infect Dis 30 (Suppl. 1):S5–S14, 2000 3. 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