The mechanism by which these HIV
The mechanism by which these HIV-PIs impair skeletal muscle palmitate transport and oxidation has been partially elucidated. CD36 (also referred to as fatty Adapalene australia translocase; FAT) is a transmembrane protein involved in the transport of long-chain fatty acids (LCFA) across cellular membranes. It has been shown that CD36 is expressed in skeletal muscle plasma membrane of C2C12 myocytes and that overexpression of this transport protein increased fatty acid uptake and utilization by myotubes . It has also been demonstrated that CD36 exists in the mitochondrial outer membrane of human skeletal muscle and was shown to participate in transport and subsequent oxidation of LCFA , . Using immunoblotting techniques, we found reduced expression of CD36 in myotubes exposed to combination HIV-PIs. It has been demonstrated that antiretroviral therapy, specifically ritonavir, nelfinavir, and the combination LPV/r, decreased expression of CD36 in HIV-infected and healthy human subject monocytes as well as in human THP-1 monocyte and human C32 melanoma cell lines . Our findings indicate that ATV/r, LPV/r, and DRV/r reduce CD36 protein expression in C2C12 myotubes by 16%, 17%, and 21% , respectively, suggesting that this contributes to the observed decrease in palmitate oxidation (ATV/r, LPV/r, and DRV/r; 15%, 50%, and 32%, respectively). It might be suggested that these drugs may induce apoptosis of the myotubes, similar to their effects in pancreatic β-cells , however, TUNEL analyses of cells treated with the HIV-PI regimens alone or with the HIV-PI regimens and then fatty acids revealed no incidence of TUNEL-positive cells (data not shown). These findings indicate that the concentrations and duration of treatments used in the present studies do not induce apoptosis of the murine skeletal muscle cells. Carnitine palmitoyltransferase 1 (CPT1) is a mitochondrial transport protein that traverses the outer mitochondrial membrane. Malonyl-CoA is a known intracellular antagonist of CPT1. Increased concentrations of acetyl-CoA and intracellular CO2 lead to the formation of malonyl-CoA which directly inhibits CPT1 expression and in turn, decreases fatty acid uptake into the mitochondria. It has been suggested that mitochondrial CD36 and CPT1 interact to transport acylcarnitines across the outer mitochondrial membrane to carnitine–acylcarnitine translocase (CAT), then on to CPT2, and finally to the intermitochondrial space for oxidation . Despite 16–21% reductions in CD36 protein levels among the HIV-PI combinations, only LPV/r and DRV/r significantly reduced CPT1 protein levels in myotubes. This implies that ATV/r reduces palmitate oxidation through a mechanism that differs from that of LPV/r and DRV/r. We cannot discount the possibility that LPV/r and DRV/r decreased CD36 protein levels, which reduced fatty acid availability to CPT1, and this subsequently reduced mitochondrial fatty acid uptake and oxidation by the muscle cells. It is not unexpected that the ATV/r regimen has different effects than the other regimens studied. It is well-recognized that different HIV-PIs have variable effects on cellular lipid/cholesterol metabolism and circulating lipid/lipoprotein levels , , , , . In clinical trials, ATV/r and LPV/r are well-tolerated, have equivalent virologic and immunologic potencies, but ATV/r is associated with a more favorable impact on serum lipid levels, and switching from a regimen that includes LPV/r, to one that includes ATV/r is associated with improvements in serum lipid/lipoprotein profiles. In lipid-loaded C2C12 cells (Fig. 7), CPT-1 expression was not reduced as much by ATV/r (−4%) as it was by LPV/r (−26%) or DRV/r (−28%) exposures. We suggest that LPV/r and DRV/r exposure reduce β-oxidation in C2C12 by inhibiting both palmitate uptake and CPT-1-associated β-oxidation. However, as noted for cholesterol metabolism in rat hepatocytes and macrophages , ATV/r does not disrupt fatty acid uptake or β-oxidation as much as LPV/r and DRV/r exposure. Instead, ATV/r must mediate its effects on C2C12 lipid metabolism at sites downstream from cellular uptake and CPT-1 expression. This may reflect a difference in ATV-protein binding, lipid membrane solubility, or affinity for lipid transporters among the different PIs.