Role of NFATc4 in ARV induced adipocyte toxicity

Background: HIV associated lipodystrophy (HIVLD) is an adverse effect of combination antiretroviral therapy (cART) which further introduces complexity in the management of HIV infection. There is also an increase in the risk of cardiovascular disease in HIV patients, even in the absence of HIVLD[1]. cART impairs adipogenesis and dysregulates the secretion of adipokines, fat and glucose metabolism in the adipose tissue. Both protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors have been implicated[2]. Nuclear Factors of Activated T cells (NFAT)c4 is an inducible nuclear factor which plays a role in adipogenesis; it has also been recently suggested to mediate the effects of cytokines on adiponectin(AdipoQ), a marker of insulin resistance[3]. The aim of this project was to examine the mechanism of NFATc4 in the pathogenesis of cART-induced metabolic disturbances using an in vitroadipocyte model.

Methods: Differentiating 3T3-F442A murine adipocytes were treated with ARVs before and after knock down of NFATc4 gene using siRNA. The effect of NFATc4 knockdown(KD)on AdipoQ and interleukin 6 (IL6)protein levels were assessed using ELISA; expression levels of Peroxisome proliferator-activated receptor gamma (PPARg)and NFATc4 proteins was measured using Western blot. AdipoQ and IL6 secretion levels were also assessed in preadipocytes and in adipocytes coincubated with lopinavir (LPV) and Telmisartan (TEL). Data are presented as mean+ SD for 20µM incubation of ARVs and 5µM -ofTEL.

Results: All ARVs resulted in a dose-dependent increase in gene expression level of NFATc4 (LPV, 1.9+0.05 [p=0.001]; atazanavir, 2.1+0.1 [p=0.0001]; ritonavir, 1.4+0.05 [p=0.0005] and efavirenz, 1.8+0.02 [p=0.0004]) as compared to the vehicle control; this also correlated with its protein expression levels. Co-incubation with TEL partially attenuated ARV-mediated upregulation NFATc4 (1.2+0.1 [p=0.0001]).siRNA KD of NFATc4 reversed the ARV-mediated effect on protein expression levels of AdipoQ (without NFATc4 KD, 3.6+0.3 pg/ml; with NFATc4 KD,7.5+0.4 pg/ml [p=0.008]), PPARg (without NFATc4 KD, 1.1+0.4; with NFATc4 KD 1.7+0.2; [p=0.02]) and IL6 (without NFATc4 KD, 81.6+1.2pg/ml; with NFATc4 KD, 13.168+1.3pg/ml [p=0.009]), and showed results similar to that obtained with TEL.

Conclusion: Our data suggests that ARV-induced upregulation of NFATc4 could be a mediator of ARV-induced adipocyte toxicity, the upregulation of proinflammatory cytokines and potentially insulin resistance.

References:

1. Pushpakom, S.P., et al. Pharmacogenet Genomics, 2011. 21(2): p. 76-83.

2. da Cunha, J., et al. World J Virol, 2015. 4(2): p. 56-77.

3. Yang, T.T., et al. Mol Cell Biol, 2006. 26(20): p. 7372-87.