The most notable effect of 4 weeks of lopinavir/ritonavir treatment on HIV seronegative men was the 83% increase in triglyceride levels with the greatest increase seen in VLDL particles. These results are similar to but quantitatively less than the threefold increase in triglyceride levels seen in HIV-negative subjects treated with ritonavir for 2 weeks [15
]. Ritonavir is the likely agent causing the increase in triglyceride levels in the lopinavir/ritonavir combination. The effect of lopinavir cannot be studied in isolation, as ritonavir is needed as a pharmocokinetic booster by blocking cytochrome CYP 3A4; therefore, one cannot determine whether lopinavir alone contributes to the elevation in triglyceride levels seen in this study.
One postulated mechanism for the elevation in triglyceride levels is an increase in VLDL production. Our results support this hypothesis over a delay in lipoprotein clearance for several reasons. First, the small 14% decrease in triglyceride level clearance seen during the intravenous fat tolerance test cannot explain the nearly twofold elevation in triglyceride levels. Second, we did not see the appearance of increased IDL cholesterol levels or remnants. Finally, others have shown that lipoprotein lipase and hepatic lipase levels remained unchanged after ritonavir treatment, thus excluding the hypothesis of the decreased lipolysis of lipoproteins as a cause of triglyceride elevation [15
]. In-vitro studies have suggested several cellular mechanisms for the increase in VLDL production. Ritonavir has been shown to inhibit apolipoprotein B degradation, resulting in an increased secretion of apolipoprotein B from cultured hepatoma cells [25
]. Others have suggested the involvement of activated sterol regulatory element binding proteins (SREBP) in the liver [26
]. Further work is needed on the mechanism of increased VLDL cholesterol production induced by ritonavir and lopinavir/ritonavir.
In contrast to the effects on VLDL and triglyceride levels, lopinavir/ritonavir did not increase LDL cholesterol levels. Increases in LDL have been observed in HIV-positive patients on HAART with several PI [15
]. However, HAART with a non-nucleoside reverse transcriptase inhibitor (NNRTI), such as nevirapine, raises LDL cholesterol to similar levels as indinavir, ritonavir, nelfinavir and saquinavir [3
]. In contrast, the treatment of HIV-negative individuals with four different PI: ritonavir [15
], indinavir [7
], amprenavir [14
], and lopinavir/ritonavir (this study), did not lead to an increase in LDL levels. These data suggest that PI therapy does not directly cause alterations in LDL metabolism. Whether these changes in LDL levels seen in HIV-positive patients on PI and NNRTI represent a restoration to health, or an interaction between HAART and HIV remains to be determined. Preliminary data indicate that atazanavir does not increase LDL in HIV-positive patients [30
]. Therefore, multiple mechanisms for alterations in LDL levels may be involved.
Although lopinavir/ritonavir had marked effects on triglyceride, FFA and VLDL cholesterol metabolism, less dramatic effects were seen on glucose metabolism and insulin resistance. Unlike indinavir, lopinavir/ritonavir had no effect on fasting glucose, fasting insulin, or HOMA, an assessment of fasting insulin resistance. No difference in insulin sensitivity or the induction of insulin resistance was seen in 10 patients using the euglycemic hyperinsulinemic clamp. However, there was a small, but significant, decrease in glucose tolerance at 120 min of OGTT. A similar impairment in glucose tolerance during OGTT, but with little insulin resistance by the minimal model, was found with amprenavir treatment of HIV-positive patients [31
]. We also previously found an increase in insulin and glucose at 120 min with indinavir 4-week treatment of healthy normal volunteers [7
]. There are several potential explanations for the different effects of lopinavir/ritonavir on glucose metabolism compared with amprenavir and indinavir. First, the changes seen during the OGTT may be independent of GLUT4 blockade, and may be caused by impaired first and second-phase insulin secretion. Woerle et al
] showed in 13 HIV-infected patients starting various PI including lopinavir/ritonavir and indinavir that first-phase insulin secretion was decreased by 25% during the hyperglycemic clamp. Second-phase insulin secretion did not decrease, but was inappropriately reduced in the setting of peripheral insulin resistance, as reflected in a decreased disposition index. Lopinavir/ritonavir may thus impair both first and second-phase insulin secretion more than peripheral resistance, resulting in impaired glucose tolerance on OGTT, with little evidence of impaired insulin-mediated glucose disposal on euglycemic hyperinsulinemic clamp. The decreased insulin levels at 30 min during the OGTT are consistent with impaired first-phase insulin secretion. An effect on insulin clearance is less likely, as levels during the clamp were not affected. Alternatively, the 10% decrease induced by lopinavir/ritonavir in glucose tolerance by AUC during the OGTT is small, and it is possible that a small change may have been missed on euglycemic hyperinsulinemic clamp or fasting glucose and insulin levels. The finding that 2 h insulin levels on OGTT increased in those with the largest increase in glucose supports this hypothesis. Although insulin levels at 120 min are elevated, second-phase insulin secretion may not be adequately increased in the setting of insulin resistance. A combination of mild insulin resistance coupled with impaired secretion preventing an adequate compensatory increase in insulin is therefore likely. Although we have not performed a randomized trial of lopinavir/ritonavir versus indinavir, it is of note that on lopinavir/ritonavir one patient developed impaired glucose tolerance, whereas on indinavir, one developed diabetes and two developed impaired glucose tolerance. The clinical significance of the small impairment of glucose tolerance on lopinavir/ritonavir is uncertain.
The lack of significant insulin resistance seen with lopinavir/ritonavir treatment during the clamp differs from the current in-vitro data on PI. Indinavir, amprenavir, and ritonavir have been shown to inhibit glucose uptake in 3T3-L1 adipocytes, and indinavir has been shown to inhibit glucose uptake by the acute blockade of GLUT4 transporters in a Xenopus laevis oocyte GLUT4 expression system. Of note, the effect of lopinavir on GLUT4 in vitro remains to be studied. One consideration in comparing the in-vivo effects of indinavir with other PI is the lower protein binding of indinavir. Whereas indinavir is only 60–65% protein bound, ritonavir and lopinavir are over 98% protein bound. The in-vitro studies of PI did not use normal concentrations of serum proteins, and therefore may have had higher free drug levels that do not account for the possible in-vivo effects of protein binding seen with lopinavir and ritonavir. Indinavir may thus achieve higher serum unbound drug concentrations in vivo than other PI.
Another difference between indinavir and lopinavir/ritonavir treatment was their effects on the FFA level. Four-week treatment with indinavir did not increase FFA, whereas lopinavir/ritonavir raised FFA by 30%. Although the stimulation of lipolysis with elevated FFA could be considered a mechanism for the insulin resistance of PI, this is not likely to be the case for several reasons. First, treatment with indinavir induced more insulin resistance than lopinavir/ritonavir by euglycemic hyperinsulinemic clamp, and yet did not increase fasting FFA levels, but tended to decrease them. Second, both indinavir and lopinavir/ritonavir showed FFA suppression during the OGTT. Third, indinavir acutely induced insulin resistance during a euglycemic hyperinsulinemic clamp with normal suppression of FFA. Finally, lopinavir/ritonavir induced a robust increase in fasting FFA without inducing insulin resistance. Although some have speculated that insulin resistance in HIV infection occurs secondary to the increased release of FFA, such studies demonstrate an uncoupling of FFA release and an induction of insulin resistance.
Likewise, it might be postulated that PI-induced insulin resistance might be the cause of the observed hypertriglyceridemia after lopinavir/ritonavir treatment. However, indinavir induced insulin resistance without increasing triglyceride levels, and lopinavir/ritonavir increased triglyceride levels without inducing significant insulin resistance. These data demonstrate that the effects of the drugs on each metabolic pathway are independent. They also emphasize the need to study other PI drugs on multiple metabolic pathways.
There are several potential limitations to the current study. There were no significant changes in body composition after 4 weeks of treatment with lopinavir/ritonavir. There was a trend towards a small decrease (4%) in visceral fat by CT scanning, but no decrease in subcutaneous fat, or total fat by DEXA. We cannot rule out the possibility that this small decrease in visceral adipose tissue may have dampened the induction of insulin resistance, but it might also have dampened the increase in FFA and triglyceride levels. The lack of increase in visceral adipose tissue suggests that changes in lipid and glucose metabolism seen with lopinavir/ritonavir are independent of increased central fat accumulation.
The drug combination of lopinavir/ritonavir limits the ability to identify which of the individual drugs or whether the combination of both are the causative agents of the effects on glucose and lipid metabolism. However, the combination is currently used to treat HIV patients and may be more relevant clinically [33
]. Four-week treatment with lopinavir/ritonavir may not have been taken long enough to induce significant changes in body composition. Only men were enrolled in the current study, and pre and postmenopausal women may have different metabolic outcomes.
In summary, 4-week treatment with lopinavir/ritonavir increased plasma triglyceride levels and worsened glucose tolerance during OGTT; however, insulin sensitivity as measured by the euglycemic hyperinsulemic clamp was not impaired. These results contrast with those seen with indinavir, which had little effect on lipid metabolism but significantly increased insulin resistance. The metabolic effects of PI thus appear to be drug specific and not class specific. Individual PI need to be studied with respect to their effects on lipid and glucose metabolism in vivo and in the presence and absence of HIV. As of yet, no PI has been shown to increase LDL significantly in HIV-negative individuals. Further exploration of the mechanism of altered lipid metabolism with PI may lead to a better understanding of lipoprotein production, and long-term studies may offer a better insight into the risk of coronary artery disease in patients with HIV infection.