Therapeutic Dose of HIV-1 Protease Inhibitor Saquinavir does not Permanently Influence Early Insulin Signaling

Abstract

The introduction of HIV-1 protease inhibitor therapy has significantly improved the expectancy and quality of life for HIV-infected patients. Recent reports have highlighted the development of metabolic complications in patients taking protease inhibitors, including abnormalities in glucose metabolism such as impaired glucose tolerance and type 2 diabetes. The mechanisms by which protease inhibitors induce these metabolic syndromes are not well understood. The aim of this study was to determine whether treatment with the HIV-1 protease inhibitor, saquinavir, influences the early insulin signaling cascade in insulin-sensitive cell lines.

Methods

Insulin-stimulated phosphorylation of insulin receptor (IR-β), insulin receptor substrates (IRS-1 and IRS-2), association of phosphatidylinositol 3-kinase (PI 3-kinase), Ser⁴⁷³-phosphorylation of Akt and Thr²⁰²/Tyr²⁰⁴-phosphorylated p44/42 MAP kinase in 3T3L1 adipocytes and FAO hepatoma cells incubated with increasing concentrations of saquinavir for 24, 36 hours, 2, 3 and 6 days were measured.

Results

Phosphorylation of IR-β, IRS-1 and IRS-2 was not permanently affected by incubation with therapeutic doses (2.5 µM) of saquinavir for 36 hours. After 24 hours we observed an increase of IR-β and IRS-1 phosphorylation. However, this initial stimulation of IR-β and IRS-1 phosphorylation was not permanent and did not result in an increased PI 3-kinase association. Phosphorylation of IRS-2 and MAP kinase as well as glucose transport activity was not altered by therapeutic doses. Doses of 10, 25 and 50 µM of saquinavir altered the early insulin signaling events in a dose-dependent manner. However, this effect was primarily due to the cytotoxic effect of higher saquinavir doses. Glucose transport activity was not significantly reduced in 3T3L1 cells treated with 2.5 µM saquinavir in comparison to the control cells stimulated with insulin.

Conclusion

Early insulin signaling cascade, essential for normal glucose metabolism, is not affected by therapeutic doses of saquinavir. The reduction of insulin-induced phosphorylation in higher concentrations is primarily related to cytotoxic effects. Other mechanisms than early insulin signaling must be primarily responsible for the metabolic alterations during saquinavir therapy.

HIV-1 protease inhibitors - saquinavir - insulin signaling - diabetes type 2

References

• 1 Algenstaedt P, Antonetti D A, Yaffe M B, Kahn C R. Insulin receptor substrate proteins create a link between the tyrosine phosphorylation cascade and the Ca²⁺-ATPases in muscle and heart.  J Biol Chem. 1997;  272 23696-23702
• 2 Antonetti D A, Algenstaedt P, Kahn C R. Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.  Mol Cell Biol. 1996;  16 2195-2203
• 3 Barry M, Gibbons S, Back D, Mulcahy F. Protease inhibitors in patients with HIV disease. Clinically important pharmacokinetic considerations.  Clin Pharmacokinet. 1997;  32 194-209
• 4 Behrens G, Dejam A, Schmidt H, Balks H J, Brabant G, Korner T, Stoll M, Schmidt R E. Impaired glucose tolerance, beta cell function and lipid metabolism in HIV patients under treatment with protease inhibitors.  Aids. 1999;  13 F63-70
• 5 Burgering B M, Coffer P J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction [see comments].  Nature. 1995;  376 599-602
• 6 Caron M, Auclair M, Vigouroux C, Glorian M, Forest C, Capeau J. The HIV protease inhibitor indinavir impairs sterol regulatory element- binding protein-1 intranuclear localization, inhibits preadipocyte differentiation, and induces insulin resistance.  Diabetes. 2001;  50 1378-1388
• 7 Carr A, Samaras K, Chisholm D J, Cooper D A. Pathogenesis of HIV-1-protease inhibitor-associated peripheral lipodystrophy, hyperlipidaemia, and insulin resistance.  Lancet. 1998;  351 1881-1883
• 8 Carr A, Samaras K, Thorisdottir A, Kaufmann G R, Chisholm D J, Cooper D A. Diagnosis, prediction, and natural course of HIV-1 protease-inhibitor- associated lipodystrophy, hyperlipidaemia, and diabetes mellitus: a cohort study [see comments].  Lancet. 1999;  353 2093-2099
• 9 Cusi K, Maezono K, Osman A, Pendergrass M, Patti M E, Pratipanawatr T, DeFronzo R A, Kahn C R, Mandarino L J. Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle.  J Clin Invest. 2000;  105 311-320
• 10 Czech M P, Corvera S. Signaling mechanisms that regulate glucose transport.  J Biol Chem. 1999;  274 1865-1868
• 11 Danner S A, Carr A, Leonard J M, Lehman L M, Gudiol F, Gonzales J, Raventos A, Rubio R, Bouza E, Pintado V, Aquado A G, Garcia de Lomas J, Delgado R, Borleffs J CC, Hsu A, Boucher C AB, Cooper D A. A short-term study of the safety, pharmacokinetics, and efficacy of ritonavir, an inhibitor of HIV-1 protease. European-Australian Collaborative Ritonavir Study Group.  N Engl J Med. 1995;  333 1528-1533
• 12 Dube M P, Johnson D L, Currier J S, Leedom J M. Protease inhibitor-associated hyperglycaemia [letter].  Lancet. 1997;  350 713-714
• 13 Eastone J A, Decker C F. New-onset diabetes mellitus associated with use of protease inhibitor [letter].  Ann Intern Med. 1997;  127 948
• 14 Flexner C. HIV-protease inhibitors [see comments].  N Engl J Med. 1998;  338 1281-1292
• 15 Franke T F, Yang S I, Chan T O, Datta K, Kazlauskas A, Morrison D K, Kaplan D R, Tsichlis P N. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase.  Cell. 1995;  81 727-736
• 16 Frost S C, Lane M D. Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes.  J Biol Chem. 1985;  260 2646-2652
• 17 Kahn C R. Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes.  Diabetes. 1994;  43 1066-1084
• 18 Katagiri H, Asano T, Ishihara H, Inukai K, Shibasaki Y, Kikuchi M, Yazaki Y, Oka Y. Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3- kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes.  J Biol Chem. 1996;  271 16987-16990
• 19 Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.  Nature. 1970;  227 680-685
• 20 Lazdins J K, Mestan J, Goutte G, Walker M R, Bold G, Capraro H G, Klimkeit T. In vitro effect of alpha1-acid glycoprotein on the anti-human immunodeficiency virus (HIV) activity of the protease inhibitor CGP 61, 755: a comparative study with other relevant HIV protease inhibitors.  J Infect Dis. 1997;  175 1063-1070
• 21 Mukherjee R, Davies P J, Crombie D L, Bischoff E D, Cesario R M, Jow L, Hamann L G, Boehm M F, Mondon C E, Nadzan A M, Paterniti Jr J R, Heymann R A. Sensitization of diabetic and obese mice to insulin by retinoid X receptor agonists.  Nature. 1997;  386 407-410
• 22 Mulligan K, Grunfeld C, Tai V W, Algren H, Pang M, Chernoff D N, Lo J C, Schambelan M. Hyperlipidemia and insulin resistance are induced by protease inhibitors independent of changes in body composition in patients with HIV infection.  J Acquir Immune Defic Syndr. 2000;  23 35-43
• 23 Murata H, Hruz P W, Mueckler M. The mechanism of insulin resistance caused by HIV protease inhibitor therapy.  J Biol Chem. 2000;  275 20251-20254
• 24 Nolte L A, Yarasheski K E, Kawanaka K, Fisher J, Le N, Holloszy J O. The HIV protease inhibitor indinavir decreases insulin- and contraction- stimulated glucose transport in skeletal muscle.  Diabetes. 2001;  50 1397-1401
• 25 Ranganathan S, Kern P A. The HIV protease inhibitor saquinavir impairs lipid metabolism and glucose transport in cultured adipocytes.  J Endocrinol. 2002;  172 155-162
• 26 Rothenberg P L, Lane W S, Karasik A, Backer J, White M, Kahn C R. Purification and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor tyrosine kinase.  J Biol Chem. 1991;  266 8302-8311
• 27 Rudich A, Vanounou S, Riesenberg K, Porat M, Tirosh A, Harman-Boehm I, Greenberg A S, Schlaeffer F, Bashan N. The HIV protease inhibitor nelfinavir induces insulin resistance and increases basal lipolysis in 3T3-L1 adipocytes.  Diabetes. 2001;  50 1425-1431
• 28 Schutt M, Meier M, Meyer M, Klein J, Aries S P, Klein H H. The HIV-1 protease inhibitor indinavir impairs insulin signalling in HepG2 hepatoma cells.  Diabetologia. 2000;  43 1145-1148
• 29 Skolnik E Y, Batzer A, Li N, Lee C H, Lowenstein E, Mohammadi M, Margolis B, Schlessinger J. The function of GRB2 in linking the insulin receptor to Ras signaling pathways.  Science. 1993;  260 1953-1955
• 30 Sun X J, Miralpeix M, Myers Jr M G, Glasheen E M, Backer J M, Kahn C R, White M F. Expression and function of IRS-1 in insulin signal transmission.  J Biol Chem. 1992;  267 22662-22672
• 31 Sun X J, Wang L M, Zhang Y, Yenush L, Myers Jr M G, Glasheen E, Lane W S, Pierce J H, White M F. Role of IRS-2 in insulin and cytokine signalling.  Nature. 1995;  377 173-177
• 32 Vella S, Floridia M. Saquinavir. Clinical pharmacology and efficacy.  Clin Pharmacokinet. 1998;  34 189-201
• 33 Visnegarwala F, Krause K L, Musher D M. Severe diabetes associated with protease inhibitor therapy [letter] [see comments].  Ann Intern Med. 1997;  127 947
• 34 Walli R, Herfort O, Michl G M, Demant T, Jager H, Dieterle C, Bogner J R, Landgraf R, Goebel F D. Treatment with protease inhibitors associated with peripheral insulin resistance and impaired oral glucose tolerance in HIV-1-infected patients.  Aids. 1998;  12 F167-173