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Chapter 13. In vitro and in vivo approaches to studying antiretroviral therapy (ART)-induced metabolic complications
Chapter 13. In Vitro and In Vivo Approaches Antiretroviral Therapy (ART)-Induced Metabolic
to Studying Complications
James E. Weiel and James M. Lenhard GlaxoSmithKline 5 Moore Drive (3.2234), Research Triangle Park, North Carolina, 27709 tntroduction - HIV protease inhibitors (Pls), as a component of highly active antiretroviral therapy (HAART), are effective antiretroviral agents in the treatment of HIV/AIDS (1). These agents inhibit the HIV-1 aspartyl protease, an enzyme essentiai for virion maturation and release and have played a major role in reducing the morbidity and mortality associated previously with HIV infection (2-4). Despite their effectiveness, long-term therapy with the Pls has been associated with a syndrome or syndromes of peripheral fat wasting, fat redistribution and alterations of glucose and lipid metabolism following treatment (512). However, not all patients treated with Pls develop these metabolic complications and in those that do, the degree to which these adverse events manifest themselves, varies (9-12). Identification of factors that influence an individual’s predisposition to acquiring any or all of these adverse complications remains elusive. Nonetheless, the appearance of these abnormalities following HAART suggests that lipid metabolism in adipocytes and hepatocytes represents an ideal focal point for studies in order to ascertain their underlying cause. Thus, since the first report of HAART-associated metabolic complications, a number of groups have turned their attention to the adipocyte and various aspects of its physiology in response to treatment with Pls. Employing in vitro cell-culture or in vwo animal model systems, these studies have taken a reductionist approach to look at the effects of Pls in isolation, away from the compounding influences of the other components of the HAART regimen. As a consequence, insights into how alterations of fat metabolism, in response to PI therapy, may lead to the development of the metabolic complications, have been obtained. This review discusses these findings and their implications for designing more efficacious antiretroviral therapies with reduced potential for causing metabolic complications in subjects treated with HAART. Background - Although HAART has been key to prolonging life for HIV-infected individuals, long-term therapy in a number of patients has been associated with the development of adverse metabolic complications (5-12). Four main elements of the HIV Lipodystrophy Syndrome (HLS) have been identified and include complications of fat wasting (lipoatrophy), fat accumulation/redistribution (lipohypertrophy), and various disturbances of lipid and glucose metabolism. Lipoatrophy invariably involves loss of peripheral subcutaneous fat from the face and extremities whereas lipohypertrophy involves increases in visceral fat leading to central (truncal) obesity as well as redistribution to the dorsocervical fat pad (buffalo hump) and focal lipomatous growths in the subcutaneous compartment of the head and extremities (13). Disturbances of lipid and glucose metabolism result in increased serum levels of cholesterol (CH), triglyceride (TG), fatty acid (FA), insulin and glucose which can lead to insulin resistance and ultimately, diabetes (11,14,15). The existence of various metabolic abnormalities arising in a significant number of patients following antiretroviral therapy was first recognized following the introduction of Pls and as a result, were attributed initially to the PI arm of HAART (5-12). However, upon further analysis by multiple groups, independent associations between the appearance of the metabolic complications and various factors indicated the underlying cause of HLS to be multi-faceted and not solely the result of PI usage (1618). Numerous factors, including various aspects of HIV disease itself as well as the patient’s past and current drug regimen, are now recognized as being involved in the
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development of these abnormalities. Thus, all elements of HAART including reverse-transcriptase nucleoside inhibitors (NRTls), non-nucleoside reversetranscriptase inhibitors (NNRTls), and Pls have been shown to be culpable to varying degrees (13.16-I 8). However, a major emphasis has been placed on determining how PI usage contributes to the observed adverse reactions, in part, due to the major role that Pls play in reducing the morbidity and mortality associated with HIV infection. In recent years, a greater understanding of and appreciation for the complexity of adipose (fat) tissue metabolism has been obtained which demonstrates that this tissue has functions well beyond the mere storage of excess fuel reserves (19-21). Indeed, adipose tissue possesses an endocrine function and serves as an active participant in the metabolic process (20,21). One of the molecules secreted by adipocytes, leptin, serves as a “lipid sensor” to regulate satiety and energy expenditure (22-24). In addition, evidence suggests a role for leptin in control of adipose tissue distribution and mass (25,26). Functionally distinct depots of adipose tissue exist throughout the body with differential regulation between the depots. For example, subcutaneous fat is more responsive than visceral fat to signaling mediated by the heterodimer nuclear receptor complex formed by the peroxisome proliferator activated receptor y (PPARy) and the retinoid X receptor (RXR) (27). The PPARyIRXR pair is known to be a potent mediator of adipogenesis, a process by which mature adipocytes are formed from immature precursor cells (19,28). The fact that the various metabolic abnormalities observed following HAART are correlated closely with alterations of fat and glucose metabolism has brought studies in this arena to the forefront, in attempts to elucidate how and why these complications occur. In Vitro APPROACHES Adipocyte Biolocly - Recognizing the fundamental importance of fat metabolism, a number of studies on the effects of Pls on this process have focused on determining whether or not these agents can affect adipocyte differentiation. An initial report to address this issue in cell culture investigated the effects of ritonavir (RTV) and indinavir (IDV) on the differentiation of the murine preadipocyte cell line, 3T3-Ll (29). In culture, the differentiation of these fibroblast-like cells into mature adipocytes, a process known as adipogenesis, requires the combined action of insulin, glucocorticoids such as dexamethasone, and a CAMP-raising agent such as isobutylmethylxanthine (IBMX). Culture conditions were chosen such that either inhibitory or stimulatory effects of the As assessed by positive staining of lipid Pls on differentiation could be observed. droplets with Oil Red 0 as well as extraction and measurement of cellular TG, both RTV and IDV increased adipogenesis significantly (29). While the mechanism for the increased differentiation observed was not determined, the possibility of a release of a protease-mediated inhibition of adipogenesis by the Pls was suggested (29). In marked contrast to these results a second report, likewise using the differentiation of 3T3-Ll cells in culture as a marker of adipogenesis, demonstrated a significant impairment of this process by several Pls in a dose-wise fashion (30). Employing Oil Red 0 staining, measurements of TG concentration, and gene expression studies, adipogenesis was inhibited in the presence of either IDV, RTV, amprenavir (APV), or nelfinavir (NFV). Relative to IDV or APV, RTV and NFV had the greatest effects on inhibition of TG accumulation and expression of genes for FAbinding protein (aP2), lipoprotein lipase (LPL), and Adipo Q, marker genes for adipogenesis (28,31,32). However, the Pls did not inhibit transcriptional regulation of PPARy in transfected cells, suggesting that inhibition of adipogenesis was not the result of a direct effect of Pls on PPARy activation. A comparison of the cell culture conditions between these two reports shows them to be very similar, making it unclear as to why such disparate results were
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obtained. In partial agreement with the second report (30) a study using saquinavir (SQV) and IDV showed these Pls inhibit differentiation of human preadipocytes (33). ln this study, SQV had a greater inhibitory effect than IDV; however, neither PI was shown capable of altering expression of aP2, even when potent stimulators of both PPARy and RXR were present in culture (33). Once again it was concluded that PImediated effects on adipogenesis were occurring via a PPARylRXR-independent mechanism. Unlike murine 3T3-Ll cells or human preadipocytes, which are committed to undergo differentiation into adipocytes, murine C3HlOT1/2 cells are pluripotent mesenchymal stem cells not committed to adipocyte differentiation, yet have the capacity to do so when appropriately induced (34). Also, while insulin, glucocorticoids and IBMX induce adipogenesis of 3T3-Ll cells (35), insulin and PPARylRXR agonists induce adipogenesis of C3HlOT1/2 cells (36). Furthermore, whereas 3T3-Ll cells express a phenotype similar to white adipose tissue (35) C3HlOT1/2 cells express a phenotype similar to brown adipose tissue (36). In a study examining the affects of Pls in C3HlOT1/2 cells, NFV, RTV and SQV reduced TG accumulation, adipogenesis and expression of the adipose markers, aP2, adipsin (complement factor D) and LPL (37). Histological analysis showed NFV, RTV and SQV decreased Oil Red O-staining of cytoplasmic fat droplets and that this occurred in the presence of the RXR agonist, LGD1069, indicating the inhibitory effects were not due to an absence of RXR ligand. Furthermore, in mature adipocytes NFV, RTV and SQV increased acute lipolysis, the hydrolysis of stored TG into glycerol and FA. In contrast, APV and IDV had little effect on lipolysis, adipogenesis, or expression of aP2, adipsin and LPL in these cells (37). Although SQV inhibited ligand-binding to PPARy to a minor degree, none of the other Pls bound to the nuclear receptors RXR or PPARy, suggesting that inhibition of adipogenesis was not due to antagonism of ligand binding to this heterodimer pair. Nonetheless, the finding that NFV, RTV, and SQV inhibited expression of gene products (aP2, adipsin and LPL) under transcriptional control by PPARylRXR indicates that in some cases Pls may have an indirect effect on PPARy/RXR signaling. Impaired PPARy/RXR signaling and increased lipolysis could result in reduced differentiation of peripheral adipocytes and storage of fat. The results of these studies suggest that the effects of Pls on fat metabolism may vary between the Pls, cell types and fat depots. More recently, two additional studies have addressed further the effects of Pls on adipogenesis in 3T3-Ll cells (38,39) and as with earlier reports (29,30), seemingly contradictory results were obtained. In one study, RTV enhanced significantly 3T3-Ll preadipocyte differentiation as well as transiently raised protein expression of the mature form of adipocyte determination and differentiation factor-llsterol regulatory element-binding protein Ic (ADD-l/SREBP-lc) (38). ADD-l/SREBP-lc is a transcription factor that promotes CH biosynthesis and adipocyte differentiation (40,41). In this instance, a 30% increase in cellular TG mass was observed (38). On the other hand, employing similar culture conditions, IDV. NFV, RTV and SQV were each shown to inhibit either preadipocyte differentiation or promote adipocyte cell death to varying degrees with NFV eliciting both effects to the greatest extent (39). Furthermore, the expression of the mature form of ADD-l/SREBP-lc was reduced markedly by NFV treatment. In addition to lowered levels of TG and ADD-l/SREBP-lc expression, NFV also reduced protein expression of CCAATlenhancer-binding protein c( (C/EBPo), an additional transcription factor regulating adipogenesis (42,43), as well as PPARy and aP2. It was concluded that the reduced expression of these proteins in response to NFV was directly related to the degree of inhibition of adipogenesis observed (39). Furthermore, while treatment of mature adipocytes with either NFV, RN or SQV resulted in loss of cellular TG, effects were once again greatest with NFV and the NFV-mediated effects on cell viability were limited to mature adipocytes only and not observed in preadipocytes (39). Again, the reason for the discrepancy
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between these two reports in unclear. Perhaps, in part, it is due to subtle differences in culture conditions not yet identified. For instance, as discussed further below, differences in amount of exogenous factors, such as retinoids, may lie at the heart of such disparate results. However, perhaps the principal reason for such results lies in the emerging realization that the effects of the Pls are not all the same as distinct differences between the various members of this class to impart a spectrum of adverse events has been recognized as described above (37,39). Insulin-stimulated glucose transport into muscle and fat is a rate-limiting step in whole body glucose disposal after a meal. To determine the effects of Pls on glucose transport, studies were done that examined the effects of IDV on glucose transporters in 3T3-Ll adipocytes and transfected Xenopus oocytes (44). IDV inhibited glucose disposal by 63% at 100 PM in murine 3T3-Ll adipocytes. IDV had no effect on Glut1 and reduced Glut4 activity by 45% in Xenopus oocytes expressing either protein, suggesting that IDV may selectively inhibit the glucose transport function of Glut4 in vitro. Although it was hypothesized that this contributes to the insulin resistance in HIV-infected subjects, the effects of Pls on glucose transport in viva have not been tested. However, IDV decreases plasma insulin levels and the insulin resistance index and increases plasma glucose levels in AKR/J mice (45). This suggests IDV affects pathways other than Glut4 which are involved in glucose metabolism in viva, such as insulin secretion. Insights into a potential mechanism of action for at least one of the Pls, IDV, come from published reports showing that IDV and various retinoic acid derivatives share common toxicities including nail, skin, and hair defects (12,46,47). These reports suggest that IDV and retinoids may exert their effects through similar molecular mechanisms involving elements of the retinoid-signaling pathway. Pharmacological effects of retinoids are, in part, due to their ability to bind and transactivate heterodimers consisting of retinoid A receptors (RARs) and RXRs (48-51). At low concentrations, all trans-retinoic acid (ATRA) is selective for RAR but activates RXR at high concentrations. Whereas activation of the RAR/RXR heterodimer inhibits adipogenesis, activation of the PPARy/RXR heterodimer stimulates adipogenesis (36,512). Studies were performed to test this hypothesis in vitro by examining the effects of Pls on retinoid signaling in C3HlOT112 (53). Cells were cultured in the presence of either APV, IDV, NFV, RTV or SQV and synthetic ATRA and metabolic responsiveness assessed by measuring the activity of a retinoid-regulated protein, alkaline phosphatase (ALP) (53). Of the Pls tested, only IDV stimulated ATRAdependent ALP activity in a dose-dependent fashion and altered stem cell morphology consistent with development along an osteoblast-like lineage. While ATRA inhibits adipogenesis in these pluripotent cells, it stimulates expression of osteoblast genes, such as ALP (36.52). IDV’s effects were observed only in the presence of ATRA. Coculture with the retinoic acid receptor RAR-antagonist, AGN 193109, inhibited the synergistic effects of IDV and ATRA, implying IDV stimulates RAR signaling selectively. Significantly, when effects of IDV on adipogenesis in C3HlOT1/2 cells were reassessed following co-culture with IDV and increasing amounts of retinoic acid, marked inhibition of lipid accumulation was observed as reported previously for NFV, RN, and SQV (37). A further ramification of these findings is related to the fact that IDV had differential effects on adipogenesis using either low (25 nM) or high (400 nM) concentrations of ATRA in the presence of the PPARy agonist, BRL49653, and insulin Specifically, IDV inhibited lipid accumulation in the presence of low (53). concentrations of ATRA, but stimulated fat accumulation in the presence of high The RAR antagonist AGN 193109 did not inhibit fat concentrations of ATRA. accumulation in the presence of 400 nM ATRA and IDV, indicating the effect was not mediated by RAR, but rather by RXR. Consequently, these findings support the notion
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that altered retinoid signaling promotes some of the adverse reactions associated with lDV therapy, in particular, altered lipid metabolism. Furthermore, differential responsiveness of stem cells to low or high concentrations of ATRA is consistent with the idea that retinoids have pharmacologically distinct effects on RAR and RXR (36,4852). Lastly, these results may, in part, explain some of the discrepancies reported for the 3T3-Ll Cell line and effects of Pls on lipid metabolism discussed above if differences in actual retinoid concentrations were evident (29,30,38,39). Hepatocvte Bioloqy - Not all in vitro approaches to study the effects of Pls on lipid metabolism have focused on stem cells, preadipocytes and adipocytes. For instance, the liver plays a central role in lipoprotein metabolism and metabolic abnormalities in hepatocytes can result in deleterious consequences for the host. In spite of this, reports addressing the effects of Pls on hepatocytes are rare (5455). One study, however, employed the human hepatoma cell line, HepG2, to determine the potential for Pls to affect fat metabolism in cultured hepatocytes (54). In this study, cells were incubated with varying concentrations of Pls in the presence of [14C]-acetate and intracellular TG, FA and CH content quantified from cell lysates of PI-treated and untreated cells. NFV and Lopinavir (LPV) produced a significant concentrationdependent increase in intracellular TG synthesis (136% and 146%, respectively) at 10 PM (54). Likewise, RTV and SQV also increased significantly intracellular TG (82% and 96%, respectively) whereas APV and IDV had no effect. None of the Pls affected FA synthesis although RTV increased CH synthesis by 48%. Similar results were obtained in studies employing primary rat hepatocytes where 10 PM RN increased CH synthesis by 56&17% and RTV and NFV increased TG synthesis (167?73% and 102*52%, respectively). These results should be contrasted with those described above in adipocytes, where NFV, RTV, and SQV, but not APV or IDV (in the absence of retinoids), reduced TG synthesis and stimulated lipolysis (37). In a second paper, also employing HepG2 cells, IDV was shown to impair insulin signaling suggesting a possible association with the development of the metabolic complications (55). It remains to be determined whether or not this may alter the well known ability of insulin to suppress hepatic gluconeogenesis. The opposing effects of Pls on different cell types could help explain the loss of fat from one compartment (subcutaneous adipose tissue) with subsequent repartitioning elsewhere (liver). Increased TG synthesis in the liver by select Pls could result in increased levels of TG-laden VLDL and chylomicron particles leading to dyslipidemia and insulin resistance. However, as in adipocytes, these observations show that select Pls affect distinct metabolic pathways which could account for the differential effects of Pls observed in the clinical setting (9,11,56). When HepG2 cells were treated with NFV (10 PM) in the absence or presence of the RXR agonist, LG100268 (100 nM), or the RXR homodimer antagonist, LG100754 (100 nM), differential effects on TG synthesis resulted (54). Whereas LG100754 had no effect on TG synthesis, LG100268 or NFV increased TG synthesis significantly (72% and 145%, respectively). In cells exposed to both drugs simultaneously, cellular TG synthesis increased 335% relative to controls. Other Pls mixed with LG100268 caused similar but less pronounced effects. Furthermore, NFV and LG100268 caused differential effects on expression of mRNA for diacylglycerol acyl transferase (DGAT) and fatty acid synthase (FAS), two essential enzymes in the synthesis of TG and FA. (54). Thus, NFV increased expression of DGAT mRNA (47%, P=O.O24) to a greater extent than FAS mRNA (P=O.9, relative to controls) whereas LG100268 increased expression of FAS mRNA (57%, P
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expression studies in adipocytes mentioned above, studies such as these will be vital in elucidating the mechanisms of action of the Pls on inducing the various metabolic abnormalities manifested. These results suggest that Pls may cause dyslipidemia by altering expression of genes involved in lipid synthesis in adipocytes (29,30.37-39,53) and hepatocytes (54).
In Vivo APPROACHES Multiple genetic and environmental components influence susceptibility to metabolic diseases. For example, diet and physical activity affect the risk of developing obesity and genetic analysis reveals that Pima Indians are more susceptible to obesity than Caucasians (57). The observation that not all subjects treated with HAART develop the same metabolic complications supports the hypothesis that susceptibility to metabolic diseases varies with environment, duration of therapy, and/or genetics. Whereas select Pls may cause hyperlipidemia in humans, serum TG levels decrease in Wistar rats treated with RN and in SWRlJ mice treated with SQV or NFV (58.59). This difference in clinical and rodent studies may be due to genetic or species-specific effects or environmental factors, such as HIV infection or diet. For example, serum FA levels decrease in obesity resistant SWR/J mice but increase in obesity prone AKRlJ mice treated with NFV, indicating that genetic susceptibility to obesity influences the dyslipidemia associated with NFV (59). Similarly, serum glUCOSe levels decrease in SWR/J mice but increase in AKR/J mice fed a low fat diet and treated with IDV or NFV (59). However, RTV treatment had no significant effect on serum glucose in Wistar rats (58). Since genotypes and responses to Pls may vary between individuals in the clinic and various strains of rodents, multiple animal models are needed to understand the metabolic pathways affected by HAART. Alterations in dietary fat and carbohydrate or fasting can also influence the effects of Pls on metabolism. IDV and NFV increased blood urea nitrogen and TG levels whereas SQV increased CH levels in AKR/J mice fed a high fat, low carbohydrate diet (45). APV did not increase plasma lipid levels in these mice (45). The effects of IDV, NFV and SQV were not observed in AKR/J mice fed a low fat, high carbohydrate diet. IDV and NFV treated mice fed the latter diet had greater serum glucose levels, weight gain and fat accumulation; IDV treated mice had lower insulin; and SQV treated mice Thus, high fat, low carbohydrate diet had lower CH than placebo treated mice. increases PI-mediated hyperlipidemia while low fat, high carbohydrate diet increases PI-mediated hyperglycemia in AKR/J mice. Consistent with these observations, NFV increases serum TG in fed but not fasted AKR/J mice, whereas RTV increases glucose in fasted but not fed AKR/J mice (54). A comparison between AKR/J mice fed either low or high fat diets reveals IDV treatment increases FA, pancreatic lipase, bilirubin and alkaline phosphatase in mice fed either diet (45). NFV also increases serum FA and glycerol levels in AKR/J mice fed each diet (45). In both fasted and fed AKR/J mice, RTV treatment increases serum TG levels (54). A comparison between SWR/J and AKR/J mice reveals NFV and IDV treatment increases pancreatic tipase in both strains of mice (59). In contrast to IDV and NFV, there was little effect of SQV and APV on FA, lipase, bilirubin or alkaline phosphatase in either mouse strain (59). Taken together, these observations indicate some PI-associated affects are unaltered by diet, fasting or genetic background. The different effects of Pls in rodents and in vitro indicate that each PI has unique pharmacological properties, some which are influenced by environmental or genetic
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factors. For example, the level of serum TG levels in AKR/J mice fed a high fat diet and treated with equal amounts of Pls follows the ranking RTV>NFV=IDV>SQV=APV (455459). Several clinical reports also indicate there is variability between the Pls in their effects on hyperlipidemia. In a study of 93 patients, RTV therapy caused hyperlipidemia more than NFV or IDV therapy (56). In another study of 67 patients, NFV-treated patients had the highest median total CH. whereas RTV-treated patients had the greatest TG level (11). Similarly, patients receiving RTV or RTV/SQV had greater serum lipid levels than patients receiving IDV (5,9). These results indicate that each PI influences a distinct metabolic pathway, perhaps accounting for the differences seen for these drugs in vivo. Although the reasons for these differences remain unknown, they could result from differences in drug metabolism, proteinbinding, or cell/ tissue penetration. While the molecular target for PI-induced dyslipidemia is unclear, in viva studies indicate the Pls stimulate biochemical pathways involved in lipid uptake and TG synthesis. Treatment of AKR/J mice with IDV or NFV stimulates pancreatic lipase activity, an enzyme that hydrolyzes diglycerides in the gut and contributes to increased dietary fat absorption (45). In the clinic, post-heparin lipase activity in plasma and removal of remnant lipoproteins are unaffected by RTV, indicating that abnormal lipoprotein lipase and TG clearance do not contribute to hyperlipidemia (60). Consistent with this hypothesis, treatment of AKRlJ mice with RTV and NFV increases TG levels by 200-300% after injection with Triton WR-1339, an inhibitor of TG clearance (54). The observation that TG plasma levels increase in the absence of TG clearance indicates these Pls stimulate TG synthesis in vwo. Li
Gluconeogenesis
PI & Adipogenesis PI -1 Lipogenesis
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lipogenesis and increased absorption of dietary fat due to increased pancreatic lipase may lead to elevated levels of both VLDL- and chylomicron-triglycerides (TG), respectively. 2. Reduced expression of lipoprotein lipase (LPL) and adipsin (complement factor D) as well as decreased levels of adipogenesis and lipogenesis in subcutaneous adipose tissue results in less fat storage in this tissue. 3. Increased lipolysis of stored fat in subcutaneous adipose tissue via hormonesensitive lipase (HSL) further reduces the extent of fat storage. 4. Free fatty acids (FFA) and glycerol, the products of lipolysis, re-circulate through the liver. FFA are used as fuel or reincorporated into VLDL-khylomicron-TG while glucose is produced from glycerol via hepatic gluconeogenesis. As the syndrome progresses, serum lipids and glucose increase while adipose tissue decreases, potentially giving rise to syndromes of dyslipidemia, Insulin resistance (IR) and lipodystrophy (LD).
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Conclusion - Fat metabolism represents a focal point in attempts to identify mechanisms underlying the appearance of metabolic complications following HAART. In vitro effects of Pls on the differentiation of adipocytes and lipid metabolism In adipocytes and hepatocytes demonstrate differences in the ability of the various Pls to affect these processes in diverse cell types. Results of in vivo studies show that both genetic and environmental factors play a major role in determining the nature and extent of the complications arising. Based on the experimental results described herein, a model to illustrate how these complications may arise is presented in Figure 1. Careful analysis of the effects of infection and individual PI and NRTI on fat and carbohydrate metabolism may improve the ability to predict, prevent or treat side effects associated with HAART. Further studies on the mechanisms of action for each PI and their abilities to cause metabolic changes in the clinic are needed. The results of these studies may allow safer and more effective therapy for AIDS to be used in the future. References
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J.M. Wentworth, T.P. Burris, and V.K.K. Chatterjee, J. Endocrinol. 164 R7 (2000) J.M. Lenhard, S.A. Kliewer, M.A. Paulik, K.D. Plunket. J.M. Lehmann, and J.E Welet Biochem. Pharmacol., 54, 801 (1997). O.M. Rosen, C.J. Smith, A. Hirsch, E. Lai, and C.S. Rubin, Recent Prog. Hor. Res 5, 41,’ (1979). M.A. Paulik and J.M. Lenhard, Cell Tiss. Res., 290, 79 (1997). J.M. Lenhard, E.S. Furfine, R.G. Jain. 0. Ittoop, L.A. Qrband-Miller, S.G. Blanchard, ?$IA Paulik, and J.E. Weiel, Antiviral Res., 47, 121 (2000). A.T. Nguyen, A.M. Gagnon, J.B. Angel, and A. Sorisky, AIDS, 14.2467 (2000). P. Dowell, C. Flexner, P.O. Kwiterovich, and M.D. Lane, J. Biol. Chem., 275, 41325 (2000) X. Wang, R. Sato, MS. Brown, X. Hua, and J.L. Goldstein, Cell, 77, 53 (1994). J.B. Kim and B.M. Spiegelman. Genes Dev., l& 1096 (1996). R.J. Christy, V.W. Yang, J.M. Ntambi. D.E. Geiman, W.H. Landschulz. A.D. Friedman y Nakabeppu, T.J. Kelly, and M.D. Lane, Genes Dev., 3, 1323 (1989). R. Herrera, H.S. Ro, G.S. Robinson, K.G. Xanthopoulos. and B M Spregerman. MCI! (les’ Biol., 9, 5331 (1989). H. Murata, P.W. Hruz, and M. Mueckler. J. Biol. Chem., 275, 20251 (2000). J.M. Lenhard, D.K. Croom. J.E. Weiel, A. Spaltenstein, D.J. Reynolds, and E FurfIne. J Nutr.. 130, 2361 (2000). D. Calista and A. Boschini. Eur. J. Dermatol., lo, 292 (2000). J.O. Sass, B. Jakob-Solder. A. Heitger, G. Tzimas. and M. Sarcletti, Dermatology, 200. 40 (2000). A. Vahlquist, J. Am. Acad. Dermatol.,,a, S29 (1992). A.T. Johnson, E.S. Klein, S.J. Grlbert, L. Wang, T.K. Song, M.E. Pino, and R.A Chandraratna, J. Med. Chem., 3, 4764 (1995). K. Shudo and H. Kagechika, Adv. Drug Res., 3: 80 (1993). M.F. Boehm. L. Zhang, B.A. Badea, S.K. White, D.E. Mais, E. Berger. CM. Suto, ME Goldman, and R.A. Heyman, J.Med. Chem., 38, 3146 (1995). W. Kuri-Harcuch, Differentiation, 23, 164 (1982). J.M. Lenhard, J.E. Weiel, M.A. Paulik, and E.S. Fur-fine. Biochem. Pharmacol., 59, 1063 (2000). J.M. Lenhard, D.K. Croom. J.E. Weiel, and D.A. Winegar. Arterioscler. Thromb. Vast. BIOI 20, 2625 (2000). M. Schutt. M. Meier, M. Meyer, J. Klein, S.P. Aries, and H.H. Klein, Diabetologra, 43. 1145 (2000). D. Periard. A. Telenti, P. Sudre, J.J. Cheseaux, P. Halfon, M.J. Reymond, SM. Marcovrna. M.P. Glauser, P. Nicod, R. Darioli, and V. Mooser, Circulation 7-1 100 700 (1999). N. Cox, M. Frigge, D. Nicolae. P. Concannon, C. Hanis, G. Bell, and A. Kong, Nat. Genet.. 2, 213 (1999). J. Ye, K. Samaras, K. Bonner, G. Cooney, D. Chisholm, and E. Kraegen, AIDS, a, 2236 (1998). J. Weiel, D. Croom, E. Furfine, A. Spaltenstein, and J. Lenhard in “Seventh European Conference on Clinical Aspects and Treatment of HIV-Infection,” Monduzzl Editore Sp A Bologna, 1999, p. 31. J.Q. Purnell, A. Zambon, R.H. Knopp. D.J. Pizzuti, R. Achari. J.M. Leonard, C. Locke, and J.D. Brunzell, AIDS, 14, 51 (2000).
to Studying Complications
James E. Weiel and James M. Lenhard GlaxoSmithKline 5 Moore Drive (3.2234), Research Triangle Park, North Carolina, 27709 tntroduction - HIV protease inhibitors (Pls), as a component of highly active antiretroviral therapy (HAART), are effective antiretroviral agents in the treatment of HIV/AIDS (1). These agents inhibit the HIV-1 aspartyl protease, an enzyme essentiai for virion maturation and release and have played a major role in reducing the morbidity and mortality associated previously with HIV infection (2-4). Despite their effectiveness, long-term therapy with the Pls has been associated with a syndrome or syndromes of peripheral fat wasting, fat redistribution and alterations of glucose and lipid metabolism following treatment (512). However, not all patients treated with Pls develop these metabolic complications and in those that do, the degree to which these adverse events manifest themselves, varies (9-12). Identification of factors that influence an individual’s predisposition to acquiring any or all of these adverse complications remains elusive. Nonetheless, the appearance of these abnormalities following HAART suggests that lipid metabolism in adipocytes and hepatocytes represents an ideal focal point for studies in order to ascertain their underlying cause. Thus, since the first report of HAART-associated metabolic complications, a number of groups have turned their attention to the adipocyte and various aspects of its physiology in response to treatment with Pls. Employing in vitro cell-culture or in vwo animal model systems, these studies have taken a reductionist approach to look at the effects of Pls in isolation, away from the compounding influences of the other components of the HAART regimen. As a consequence, insights into how alterations of fat metabolism, in response to PI therapy, may lead to the development of the metabolic complications, have been obtained. This review discusses these findings and their implications for designing more efficacious antiretroviral therapies with reduced potential for causing metabolic complications in subjects treated with HAART. Background - Although HAART has been key to prolonging life for HIV-infected individuals, long-term therapy in a number of patients has been associated with the development of adverse metabolic complications (5-12). Four main elements of the HIV Lipodystrophy Syndrome (HLS) have been identified and include complications of fat wasting (lipoatrophy), fat accumulation/redistribution (lipohypertrophy), and various disturbances of lipid and glucose metabolism. Lipoatrophy invariably involves loss of peripheral subcutaneous fat from the face and extremities whereas lipohypertrophy involves increases in visceral fat leading to central (truncal) obesity as well as redistribution to the dorsocervical fat pad (buffalo hump) and focal lipomatous growths in the subcutaneous compartment of the head and extremities (13). Disturbances of lipid and glucose metabolism result in increased serum levels of cholesterol (CH), triglyceride (TG), fatty acid (FA), insulin and glucose which can lead to insulin resistance and ultimately, diabetes (11,14,15). The existence of various metabolic abnormalities arising in a significant number of patients following antiretroviral therapy was first recognized following the introduction of Pls and as a result, were attributed initially to the PI arm of HAART (5-12). However, upon further analysis by multiple groups, independent associations between the appearance of the metabolic complications and various factors indicated the underlying cause of HLS to be multi-faceted and not solely the result of PI usage (1618). Numerous factors, including various aspects of HIV disease itself as well as the patient’s past and current drug regimen, are now recognized as being involved in the
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development of these abnormalities. Thus, all elements of HAART including reverse-transcriptase nucleoside inhibitors (NRTls), non-nucleoside reversetranscriptase inhibitors (NNRTls), and Pls have been shown to be culpable to varying degrees (13.16-I 8). However, a major emphasis has been placed on determining how PI usage contributes to the observed adverse reactions, in part, due to the major role that Pls play in reducing the morbidity and mortality associated with HIV infection. In recent years, a greater understanding of and appreciation for the complexity of adipose (fat) tissue metabolism has been obtained which demonstrates that this tissue has functions well beyond the mere storage of excess fuel reserves (19-21). Indeed, adipose tissue possesses an endocrine function and serves as an active participant in the metabolic process (20,21). One of the molecules secreted by adipocytes, leptin, serves as a “lipid sensor” to regulate satiety and energy expenditure (22-24). In addition, evidence suggests a role for leptin in control of adipose tissue distribution and mass (25,26). Functionally distinct depots of adipose tissue exist throughout the body with differential regulation between the depots. For example, subcutaneous fat is more responsive than visceral fat to signaling mediated by the heterodimer nuclear receptor complex formed by the peroxisome proliferator activated receptor y (PPARy) and the retinoid X receptor (RXR) (27). The PPARyIRXR pair is known to be a potent mediator of adipogenesis, a process by which mature adipocytes are formed from immature precursor cells (19,28). The fact that the various metabolic abnormalities observed following HAART are correlated closely with alterations of fat and glucose metabolism has brought studies in this arena to the forefront, in attempts to elucidate how and why these complications occur. In Vitro APPROACHES Adipocyte Biolocly - Recognizing the fundamental importance of fat metabolism, a number of studies on the effects of Pls on this process have focused on determining whether or not these agents can affect adipocyte differentiation. An initial report to address this issue in cell culture investigated the effects of ritonavir (RTV) and indinavir (IDV) on the differentiation of the murine preadipocyte cell line, 3T3-Ll (29). In culture, the differentiation of these fibroblast-like cells into mature adipocytes, a process known as adipogenesis, requires the combined action of insulin, glucocorticoids such as dexamethasone, and a CAMP-raising agent such as isobutylmethylxanthine (IBMX). Culture conditions were chosen such that either inhibitory or stimulatory effects of the As assessed by positive staining of lipid Pls on differentiation could be observed. droplets with Oil Red 0 as well as extraction and measurement of cellular TG, both RTV and IDV increased adipogenesis significantly (29). While the mechanism for the increased differentiation observed was not determined, the possibility of a release of a protease-mediated inhibition of adipogenesis by the Pls was suggested (29). In marked contrast to these results a second report, likewise using the differentiation of 3T3-Ll cells in culture as a marker of adipogenesis, demonstrated a significant impairment of this process by several Pls in a dose-wise fashion (30). Employing Oil Red 0 staining, measurements of TG concentration, and gene expression studies, adipogenesis was inhibited in the presence of either IDV, RTV, amprenavir (APV), or nelfinavir (NFV). Relative to IDV or APV, RTV and NFV had the greatest effects on inhibition of TG accumulation and expression of genes for FAbinding protein (aP2), lipoprotein lipase (LPL), and Adipo Q, marker genes for adipogenesis (28,31,32). However, the Pls did not inhibit transcriptional regulation of PPARy in transfected cells, suggesting that inhibition of adipogenesis was not the result of a direct effect of Pls on PPARy activation. A comparison of the cell culture conditions between these two reports shows them to be very similar, making it unclear as to why such disparate results were
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obtained. In partial agreement with the second report (30) a study using saquinavir (SQV) and IDV showed these Pls inhibit differentiation of human preadipocytes (33). ln this study, SQV had a greater inhibitory effect than IDV; however, neither PI was shown capable of altering expression of aP2, even when potent stimulators of both PPARy and RXR were present in culture (33). Once again it was concluded that PImediated effects on adipogenesis were occurring via a PPARylRXR-independent mechanism. Unlike murine 3T3-Ll cells or human preadipocytes, which are committed to undergo differentiation into adipocytes, murine C3HlOT1/2 cells are pluripotent mesenchymal stem cells not committed to adipocyte differentiation, yet have the capacity to do so when appropriately induced (34). Also, while insulin, glucocorticoids and IBMX induce adipogenesis of 3T3-Ll cells (35), insulin and PPARylRXR agonists induce adipogenesis of C3HlOT1/2 cells (36). Furthermore, whereas 3T3-Ll cells express a phenotype similar to white adipose tissue (35) C3HlOT1/2 cells express a phenotype similar to brown adipose tissue (36). In a study examining the affects of Pls in C3HlOT1/2 cells, NFV, RTV and SQV reduced TG accumulation, adipogenesis and expression of the adipose markers, aP2, adipsin (complement factor D) and LPL (37). Histological analysis showed NFV, RTV and SQV decreased Oil Red O-staining of cytoplasmic fat droplets and that this occurred in the presence of the RXR agonist, LGD1069, indicating the inhibitory effects were not due to an absence of RXR ligand. Furthermore, in mature adipocytes NFV, RTV and SQV increased acute lipolysis, the hydrolysis of stored TG into glycerol and FA. In contrast, APV and IDV had little effect on lipolysis, adipogenesis, or expression of aP2, adipsin and LPL in these cells (37). Although SQV inhibited ligand-binding to PPARy to a minor degree, none of the other Pls bound to the nuclear receptors RXR or PPARy, suggesting that inhibition of adipogenesis was not due to antagonism of ligand binding to this heterodimer pair. Nonetheless, the finding that NFV, RTV, and SQV inhibited expression of gene products (aP2, adipsin and LPL) under transcriptional control by PPARylRXR indicates that in some cases Pls may have an indirect effect on PPARy/RXR signaling. Impaired PPARy/RXR signaling and increased lipolysis could result in reduced differentiation of peripheral adipocytes and storage of fat. The results of these studies suggest that the effects of Pls on fat metabolism may vary between the Pls, cell types and fat depots. More recently, two additional studies have addressed further the effects of Pls on adipogenesis in 3T3-Ll cells (38,39) and as with earlier reports (29,30), seemingly contradictory results were obtained. In one study, RTV enhanced significantly 3T3-Ll preadipocyte differentiation as well as transiently raised protein expression of the mature form of adipocyte determination and differentiation factor-llsterol regulatory element-binding protein Ic (ADD-l/SREBP-lc) (38). ADD-l/SREBP-lc is a transcription factor that promotes CH biosynthesis and adipocyte differentiation (40,41). In this instance, a 30% increase in cellular TG mass was observed (38). On the other hand, employing similar culture conditions, IDV. NFV, RTV and SQV were each shown to inhibit either preadipocyte differentiation or promote adipocyte cell death to varying degrees with NFV eliciting both effects to the greatest extent (39). Furthermore, the expression of the mature form of ADD-l/SREBP-lc was reduced markedly by NFV treatment. In addition to lowered levels of TG and ADD-l/SREBP-lc expression, NFV also reduced protein expression of CCAATlenhancer-binding protein c( (C/EBPo), an additional transcription factor regulating adipogenesis (42,43), as well as PPARy and aP2. It was concluded that the reduced expression of these proteins in response to NFV was directly related to the degree of inhibition of adipogenesis observed (39). Furthermore, while treatment of mature adipocytes with either NFV, RN or SQV resulted in loss of cellular TG, effects were once again greatest with NFV and the NFV-mediated effects on cell viability were limited to mature adipocytes only and not observed in preadipocytes (39). Again, the reason for the discrepancy
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between these two reports in unclear. Perhaps, in part, it is due to subtle differences in culture conditions not yet identified. For instance, as discussed further below, differences in amount of exogenous factors, such as retinoids, may lie at the heart of such disparate results. However, perhaps the principal reason for such results lies in the emerging realization that the effects of the Pls are not all the same as distinct differences between the various members of this class to impart a spectrum of adverse events has been recognized as described above (37,39). Insulin-stimulated glucose transport into muscle and fat is a rate-limiting step in whole body glucose disposal after a meal. To determine the effects of Pls on glucose transport, studies were done that examined the effects of IDV on glucose transporters in 3T3-Ll adipocytes and transfected Xenopus oocytes (44). IDV inhibited glucose disposal by 63% at 100 PM in murine 3T3-Ll adipocytes. IDV had no effect on Glut1 and reduced Glut4 activity by 45% in Xenopus oocytes expressing either protein, suggesting that IDV may selectively inhibit the glucose transport function of Glut4 in vitro. Although it was hypothesized that this contributes to the insulin resistance in HIV-infected subjects, the effects of Pls on glucose transport in viva have not been tested. However, IDV decreases plasma insulin levels and the insulin resistance index and increases plasma glucose levels in AKR/J mice (45). This suggests IDV affects pathways other than Glut4 which are involved in glucose metabolism in viva, such as insulin secretion. Insights into a potential mechanism of action for at least one of the Pls, IDV, come from published reports showing that IDV and various retinoic acid derivatives share common toxicities including nail, skin, and hair defects (12,46,47). These reports suggest that IDV and retinoids may exert their effects through similar molecular mechanisms involving elements of the retinoid-signaling pathway. Pharmacological effects of retinoids are, in part, due to their ability to bind and transactivate heterodimers consisting of retinoid A receptors (RARs) and RXRs (48-51). At low concentrations, all trans-retinoic acid (ATRA) is selective for RAR but activates RXR at high concentrations. Whereas activation of the RAR/RXR heterodimer inhibits adipogenesis, activation of the PPARy/RXR heterodimer stimulates adipogenesis (36,512). Studies were performed to test this hypothesis in vitro by examining the effects of Pls on retinoid signaling in C3HlOT112 (53). Cells were cultured in the presence of either APV, IDV, NFV, RTV or SQV and synthetic ATRA and metabolic responsiveness assessed by measuring the activity of a retinoid-regulated protein, alkaline phosphatase (ALP) (53). Of the Pls tested, only IDV stimulated ATRAdependent ALP activity in a dose-dependent fashion and altered stem cell morphology consistent with development along an osteoblast-like lineage. While ATRA inhibits adipogenesis in these pluripotent cells, it stimulates expression of osteoblast genes, such as ALP (36.52). IDV’s effects were observed only in the presence of ATRA. Coculture with the retinoic acid receptor RAR-antagonist, AGN 193109, inhibited the synergistic effects of IDV and ATRA, implying IDV stimulates RAR signaling selectively. Significantly, when effects of IDV on adipogenesis in C3HlOT1/2 cells were reassessed following co-culture with IDV and increasing amounts of retinoic acid, marked inhibition of lipid accumulation was observed as reported previously for NFV, RN, and SQV (37). A further ramification of these findings is related to the fact that IDV had differential effects on adipogenesis using either low (25 nM) or high (400 nM) concentrations of ATRA in the presence of the PPARy agonist, BRL49653, and insulin Specifically, IDV inhibited lipid accumulation in the presence of low (53). concentrations of ATRA, but stimulated fat accumulation in the presence of high The RAR antagonist AGN 193109 did not inhibit fat concentrations of ATRA. accumulation in the presence of 400 nM ATRA and IDV, indicating the effect was not mediated by RAR, but rather by RXR. Consequently, these findings support the notion
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that altered retinoid signaling promotes some of the adverse reactions associated with lDV therapy, in particular, altered lipid metabolism. Furthermore, differential responsiveness of stem cells to low or high concentrations of ATRA is consistent with the idea that retinoids have pharmacologically distinct effects on RAR and RXR (36,4852). Lastly, these results may, in part, explain some of the discrepancies reported for the 3T3-Ll Cell line and effects of Pls on lipid metabolism discussed above if differences in actual retinoid concentrations were evident (29,30,38,39). Hepatocvte Bioloqy - Not all in vitro approaches to study the effects of Pls on lipid metabolism have focused on stem cells, preadipocytes and adipocytes. For instance, the liver plays a central role in lipoprotein metabolism and metabolic abnormalities in hepatocytes can result in deleterious consequences for the host. In spite of this, reports addressing the effects of Pls on hepatocytes are rare (5455). One study, however, employed the human hepatoma cell line, HepG2, to determine the potential for Pls to affect fat metabolism in cultured hepatocytes (54). In this study, cells were incubated with varying concentrations of Pls in the presence of [14C]-acetate and intracellular TG, FA and CH content quantified from cell lysates of PI-treated and untreated cells. NFV and Lopinavir (LPV) produced a significant concentrationdependent increase in intracellular TG synthesis (136% and 146%, respectively) at 10 PM (54). Likewise, RTV and SQV also increased significantly intracellular TG (82% and 96%, respectively) whereas APV and IDV had no effect. None of the Pls affected FA synthesis although RTV increased CH synthesis by 48%. Similar results were obtained in studies employing primary rat hepatocytes where 10 PM RN increased CH synthesis by 56&17% and RTV and NFV increased TG synthesis (167?73% and 102*52%, respectively). These results should be contrasted with those described above in adipocytes, where NFV, RTV, and SQV, but not APV or IDV (in the absence of retinoids), reduced TG synthesis and stimulated lipolysis (37). In a second paper, also employing HepG2 cells, IDV was shown to impair insulin signaling suggesting a possible association with the development of the metabolic complications (55). It remains to be determined whether or not this may alter the well known ability of insulin to suppress hepatic gluconeogenesis. The opposing effects of Pls on different cell types could help explain the loss of fat from one compartment (subcutaneous adipose tissue) with subsequent repartitioning elsewhere (liver). Increased TG synthesis in the liver by select Pls could result in increased levels of TG-laden VLDL and chylomicron particles leading to dyslipidemia and insulin resistance. However, as in adipocytes, these observations show that select Pls affect distinct metabolic pathways which could account for the differential effects of Pls observed in the clinical setting (9,11,56). When HepG2 cells were treated with NFV (10 PM) in the absence or presence of the RXR agonist, LG100268 (100 nM), or the RXR homodimer antagonist, LG100754 (100 nM), differential effects on TG synthesis resulted (54). Whereas LG100754 had no effect on TG synthesis, LG100268 or NFV increased TG synthesis significantly (72% and 145%, respectively). In cells exposed to both drugs simultaneously, cellular TG synthesis increased 335% relative to controls. Other Pls mixed with LG100268 caused similar but less pronounced effects. Furthermore, NFV and LG100268 caused differential effects on expression of mRNA for diacylglycerol acyl transferase (DGAT) and fatty acid synthase (FAS), two essential enzymes in the synthesis of TG and FA. (54). Thus, NFV increased expression of DGAT mRNA (47%, P=O.O24) to a greater extent than FAS mRNA (P=O.9, relative to controls) whereas LG100268 increased expression of FAS mRNA (57%, P
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expression studies in adipocytes mentioned above, studies such as these will be vital in elucidating the mechanisms of action of the Pls on inducing the various metabolic abnormalities manifested. These results suggest that Pls may cause dyslipidemia by altering expression of genes involved in lipid synthesis in adipocytes (29,30.37-39,53) and hepatocytes (54).
In Vivo APPROACHES Multiple genetic and environmental components influence susceptibility to metabolic diseases. For example, diet and physical activity affect the risk of developing obesity and genetic analysis reveals that Pima Indians are more susceptible to obesity than Caucasians (57). The observation that not all subjects treated with HAART develop the same metabolic complications supports the hypothesis that susceptibility to metabolic diseases varies with environment, duration of therapy, and/or genetics. Whereas select Pls may cause hyperlipidemia in humans, serum TG levels decrease in Wistar rats treated with RN and in SWRlJ mice treated with SQV or NFV (58.59). This difference in clinical and rodent studies may be due to genetic or species-specific effects or environmental factors, such as HIV infection or diet. For example, serum FA levels decrease in obesity resistant SWR/J mice but increase in obesity prone AKRlJ mice treated with NFV, indicating that genetic susceptibility to obesity influences the dyslipidemia associated with NFV (59). Similarly, serum glUCOSe levels decrease in SWR/J mice but increase in AKR/J mice fed a low fat diet and treated with IDV or NFV (59). However, RTV treatment had no significant effect on serum glucose in Wistar rats (58). Since genotypes and responses to Pls may vary between individuals in the clinic and various strains of rodents, multiple animal models are needed to understand the metabolic pathways affected by HAART. Alterations in dietary fat and carbohydrate or fasting can also influence the effects of Pls on metabolism. IDV and NFV increased blood urea nitrogen and TG levels whereas SQV increased CH levels in AKR/J mice fed a high fat, low carbohydrate diet (45). APV did not increase plasma lipid levels in these mice (45). The effects of IDV, NFV and SQV were not observed in AKR/J mice fed a low fat, high carbohydrate diet. IDV and NFV treated mice fed the latter diet had greater serum glucose levels, weight gain and fat accumulation; IDV treated mice had lower insulin; and SQV treated mice Thus, high fat, low carbohydrate diet had lower CH than placebo treated mice. increases PI-mediated hyperlipidemia while low fat, high carbohydrate diet increases PI-mediated hyperglycemia in AKR/J mice. Consistent with these observations, NFV increases serum TG in fed but not fasted AKR/J mice, whereas RTV increases glucose in fasted but not fed AKR/J mice (54). A comparison between AKR/J mice fed either low or high fat diets reveals IDV treatment increases FA, pancreatic lipase, bilirubin and alkaline phosphatase in mice fed either diet (45). NFV also increases serum FA and glycerol levels in AKR/J mice fed each diet (45). In both fasted and fed AKR/J mice, RTV treatment increases serum TG levels (54). A comparison between SWR/J and AKR/J mice reveals NFV and IDV treatment increases pancreatic tipase in both strains of mice (59). In contrast to IDV and NFV, there was little effect of SQV and APV on FA, lipase, bilirubin or alkaline phosphatase in either mouse strain (59). Taken together, these observations indicate some PI-associated affects are unaltered by diet, fasting or genetic background. The different effects of Pls in rodents and in vitro indicate that each PI has unique pharmacological properties, some which are influenced by environmental or genetic
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factors. For example, the level of serum TG levels in AKR/J mice fed a high fat diet and treated with equal amounts of Pls follows the ranking RTV>NFV=IDV>SQV=APV (455459). Several clinical reports also indicate there is variability between the Pls in their effects on hyperlipidemia. In a study of 93 patients, RTV therapy caused hyperlipidemia more than NFV or IDV therapy (56). In another study of 67 patients, NFV-treated patients had the highest median total CH. whereas RTV-treated patients had the greatest TG level (11). Similarly, patients receiving RTV or RTV/SQV had greater serum lipid levels than patients receiving IDV (5,9). These results indicate that each PI influences a distinct metabolic pathway, perhaps accounting for the differences seen for these drugs in vivo. Although the reasons for these differences remain unknown, they could result from differences in drug metabolism, proteinbinding, or cell/ tissue penetration. While the molecular target for PI-induced dyslipidemia is unclear, in viva studies indicate the Pls stimulate biochemical pathways involved in lipid uptake and TG synthesis. Treatment of AKR/J mice with IDV or NFV stimulates pancreatic lipase activity, an enzyme that hydrolyzes diglycerides in the gut and contributes to increased dietary fat absorption (45). In the clinic, post-heparin lipase activity in plasma and removal of remnant lipoproteins are unaffected by RTV, indicating that abnormal lipoprotein lipase and TG clearance do not contribute to hyperlipidemia (60). Consistent with this hypothesis, treatment of AKRlJ mice with RTV and NFV increases TG levels by 200-300% after injection with Triton WR-1339, an inhibitor of TG clearance (54). The observation that TG plasma levels increase in the absence of TG clearance indicates these Pls stimulate TG synthesis in vwo. Li
Gluconeogenesis
PI & Adipogenesis PI -1 Lipogenesis
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1.
Increased hepatrc
lipogenesis and increased absorption of dietary fat due to increased pancreatic lipase may lead to elevated levels of both VLDL- and chylomicron-triglycerides (TG), respectively. 2. Reduced expression of lipoprotein lipase (LPL) and adipsin (complement factor D) as well as decreased levels of adipogenesis and lipogenesis in subcutaneous adipose tissue results in less fat storage in this tissue. 3. Increased lipolysis of stored fat in subcutaneous adipose tissue via hormonesensitive lipase (HSL) further reduces the extent of fat storage. 4. Free fatty acids (FFA) and glycerol, the products of lipolysis, re-circulate through the liver. FFA are used as fuel or reincorporated into VLDL-khylomicron-TG while glucose is produced from glycerol via hepatic gluconeogenesis. As the syndrome progresses, serum lipids and glucose increase while adipose tissue decreases, potentially giving rise to syndromes of dyslipidemia, Insulin resistance (IR) and lipodystrophy (LD).
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Conclusion - Fat metabolism represents a focal point in attempts to identify mechanisms underlying the appearance of metabolic complications following HAART. In vitro effects of Pls on the differentiation of adipocytes and lipid metabolism In adipocytes and hepatocytes demonstrate differences in the ability of the various Pls to affect these processes in diverse cell types. Results of in vivo studies show that both genetic and environmental factors play a major role in determining the nature and extent of the complications arising. Based on the experimental results described herein, a model to illustrate how these complications may arise is presented in Figure 1. Careful analysis of the effects of infection and individual PI and NRTI on fat and carbohydrate metabolism may improve the ability to predict, prevent or treat side effects associated with HAART. Further studies on the mechanisms of action for each PI and their abilities to cause metabolic changes in the clinic are needed. The results of these studies may allow safer and more effective therapy for AIDS to be used in the future. References
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