After a 12 h fast as well as during the hyperinsulinaemic clamp, glycerol turnover did not change in the LPV/r+NVP group over the course of 3 months (Table 3). In contrast, in the LPV/r+ZDV/3TC group, fasting glycerol turnover had significantly increased, by 22%, 3 months after starting therapy (P < 0.005). During hyperinsulinaemia, however, glycerol turnover was suppressed equally at month 3 as at baseline in the LPV/r+ZDV/3TC arm. In agreement with the enhanced glycerol turnover after an overnight fast, fasting plasma FFA levels were 84% higher in the LPV/r+ZDV/3TC group after 3 months (P < 0.001). In the LPV/r+NVP group, fasting FFA did not change over time. As a result, the change in fasting FFA was significantly different when comparing the treatment arms (P < 0.05). Additionally, after 3 months of treatment with LPV/r+ZDV/3TC, FFA concentrations were slightly but significantly higher during the clamp (P < 0.05). There was no significant correlation between the change in insulin-mediated glucose disposal and the change in fasting glycerol turnover (r = 0.13).
There were no significant differences in fasting plasma concentrations of cortisol or epinephrine over time in either treatment group (Table 2). In the LPV/r+NVP group, fasting plasma glucagon significantly decreased (P < 0.05), whereas norepinephrine levels decreased in the LPV/r+ZDV/3TC group after starting treatment (P < 0.05). Plasma adiponectin concentrations did not change significantly after 3 months of therapy. Concentrations of sTNFR-1 and sTNF-2 decreased in both arms, but the declines were only significant in the LPV/r+ZDV/3TC arm (P < 0.005 and P < 0.001, respectively). Plasma total cholesterol, high density lipoprotein (HDL)-cholesterol and triglycerides were significantly higher after treatment in both groups (P < 0.05) with no significant differences between groups.
Dyslipidaemia, body fat redistribution and disturbed glucose homeostasis are frequently seen in HIV-1-infected patients treated with cART [3,7]. Once patients have developed clinically overt signs of lipodystrophy, the disturbances in glucose metabolism have been shown to include insulin resistance at the level of peripheral glucose disposal, hepatic glucose production and lipolysis [5,6,9,35]. The present study, to our knowledge, is the first prospective randomized clinical trial that attempts to investigate the onset of the derangements in glucose kinetics and body composition in relation to commencing treatment with different cART regimens, with and without NRTI, in HIV-1-infected, ART-naive patients, by conducting hyperinsulinaemic–euglycaemic clamps. Our results show that treatment with a NRTI-containing regimen of LPV/r+ZDV/3TC, in the absence of significant changes in body fat distribution, led to a 25% decrease in insulin-mediated peripheral glucose disposal and a 22% increase in fasting lipolysis after as little as 3 months of therapy, while this was not the case in patients randomized to a NRTI-sparing regimen of LPV/r+NVP.
Results from detailed prospective studies on the metabolic side-effects of ART in HIV-1-infected patients are limited. Only a few studies have tried to investigate the effects of the PI class on insulin sensitivity, measured by intravenous glucose tolerance test, the homeostatic model assessment (HOMA) index and the hyperglycaemic clamp. These studies showed that insulin resistance was detectable only a few months after starting a PI-containing regimen in HIV-1-infected patients [10,14,22]. Since these studies contained a mixture of ART-naive and ART-experienced patients and the regimens included NRTI drugs, it is impossible to conclude whether the observed reduction in insulin sensitivity could be attributed only to the use of a PI. The effect of different PI on glucose metabolism has been studied in HIV-1-uninfected volunteers. Administration of the PI indinavir, both as a single dose and during 4 weeks, reduced peripheral glucose disposal and increased endogenous glucose production during a hyperinsulinaemic clamp [16,17,19]. The effects observed with short-term exposure to LPV/r differ however; insulin sensitivity with respect to glucose disposal decreased after a single dose and after 5 days of exposure [13,18], but not after 4 weeks of LPV/r treatment . In line with these human studies, studies in healthy rodents showed an acute reduction in peripheral glucose uptake after administration of several PI drugs, including LPV/r [12,23]. Moreover, these studies showed that glucose transport in both skeletal muscle and adipose tissue was affected. In-vitro research has shown that several PI, including ritonavir, are able to acutely inhibit the activity of glucose transporter-4 [11,15], thereby offering a possible explanation for the peripheral insulin resistance found in healthy volunteers and HIV-1-infected patients with lipodystrophy. Besides PI, NRTI drugs have also been associated with insulin resistance in patients with HIV-1-infection. Duration of exposure to NRTI has been reported to be independently associated with insulin resistance, as measured by fasting insulin levels and the QUICKI index in a number of observational studies [26,37]. These findings could potentially be explained as indirect effects of NRTI by virtue of their association with the development of changes in body fat distribution, especially lipoatrophy [24,25]. However, evidence from a randomized trial conducted in previously ART-naive subjects demonstrated early and sustained increases in fasting serum insulin levels and in HOMA indices, as short as 4 weeks after treatment initiation with cART containing a thymidine analogue, including stavudine plus didanosine, but not with a thymidine analogue-sparing combination of abacavir plus 3TC . These results suggested that thymidine analogue NRTI, in particular, may contribute to the early onset of insulin resistance before any clinically noticeable changes in body fat distribution are present. A recent placebo-controlled study in healthy HIV-uninfected volunteers demonstrated that administration of stavudine during 4 weeks indeed resulted in a significant decrease in insulin sensitivity measured by hyperinsulinaemic–euglycaemic clamps, without changes in body composition. Interestingly, 31P magnetic resonance spectroscopy revealed reduced mitochondrial function in skeletal muscle, which correlated significantly with insulin sensitivity. In addition, mitochondrial DNA content was reduced in muscle biopsies of stavudine recipients .
The present study which employed serial hyperinsulinaemic–euglycaemic clamps, found a reduction in peripheral insulin sensitivity study in the absence of significant changes in body fat distribution, after only 3 months in patients taking LPV/r+ZDV/3TC, as opposed to those treated with LPV/r+NVP. The latter may seem in contrast with the findings from earlier studies assessing changes in insulin sensitivity following the introduction of PI in antiretroviral regimens [10,14,22]. Of note however, the PI in those studies did not include LPV/r. Furthermore, patients were concomitantly receiving an NRTI in most instances, which could have contributed to the reported changes in glucose metabolism. Nonetheless, LPV/r did result in a change in insulin sensitivity when administered as monotherapy to healthy HIV-1-uninfected persons [13,18]. The difference in duration of LPV/r exposure between our study (3 months) and the studies conducted in healthy volunteers (single dose and 5 days) may be relevant and an earlier reduction in insulin sensitivity in our patients on LPV/r+NVP, which had disappeared after 3 months, cannot be ruled out.
The changes in glucose metabolism in the NRTI-containing arm, as opposed to the NRTI-sparing arm, suggest that exposure to ZDV/3TC may have contributed to the development of alterations in glucose metabolism. This is in accordance with a number of observations concerning the potential contribution of NRTI to the development of insulin resistance [26,27,37]. NRTI, particularly the thymidine analogues stavudine and ZDV, have been shown to play an important role in the onset of lipoatrophy . Since lipoatrophy has independently been associated with insulin resistance, NRTI drugs have been suggested to affect glucose metabolism indirectly via a decrease in peripheral fat [24,25]. In the present study, however, LPV/r+ZDV/3TC treatment resulted in disturbed glucose metabolism in the absence of changes in fat distribution. We cannot rule out the possibility of having missed a small decline in peripheral fat because of a concomitant increase in fat through recovery from HIV-wasting, as has been described before in patients starting ART [40,41], but find it an unlikely confounding factor as patients with wasting were excluded and LPV/r+NVP treatment, which is not expected to result in lipoatrophy, likewise did not result in increased fat amounts. This suggests that ZDV/3TC may have directly contributed to the development of reduced insulin sensitivity, through several possible mechanisms. Some NRTI drugs have been shown to disturb the production and secretion of adipocytokines, which, in turn, could affect glucose metabolism. In 3T3 culture adipocytes, stavudine and ZDV both decreased adiponectin production, whereas the production of TNF-α and interleukins 1 and 6 was enhanced . However, in the present study, adiponectin levels were unchanged after 3 months, whereas sTNFR-1 and sTNF-2 decreased rather than increased in the LPV/r+ZDV/3TC arm. These results argue against abnormal adipocytokine secretion as an explanation for insulin insensitivity. In our study, LPV/r+ZDV/3TC treatment resulted in stimulation of fasting lipolysis with increased plasma FFA levels, which may indicate resistance to insulin's suppressive effect on lipolysis. As FFA have a negative influence on the signalling pathway of insulin [43,44], increased FFA levels could have contributed to insulin resistance with respect to peripheral glucose disposal in the LPV/r+ZDV/3TC arm. However, since patients with HIV-associated lipodystrophy remained insulin resistant (albeit less so) after FFA levels were acutely lowered by administration of the lipolysis inhibitor acipimox , additional factors are involved. There might be an important role for mitochondria in fat and muscle in the development of insulin insensitivity. NRTI have been associated with mitochondrial toxicity [38,46–49]. A decline in oxidative phosphorylation, resulting from mitochondrial dysfunction, could result in accumulation of FFA (metabolites), which, in turn, could have a negative effect on the signalling cascade of insulin [43,44] and may reinforce mitochondrial dysfunction [44,50]. NRTI-induced mitochondrial dysfunction as a cause for insulin insensitivity has been suggested by a recent study in healthy volunteers .
With respect to plasma lipids, total cholesterol and triglyceride levels increased significantly in both arms after 3 months, which was not unexpected given the presence of LPV/r in both regimens [51,52]. Compatible with observations in other trials of potent cART in ART-naive, HIV-1-infected individuals, HDL-cholesterol likewise increased in both arms [53,54]. Further follow-up of lipid changes in all MEDICLAS trial participants will be needed to determine whether the rise in HDL-cholesterol will be significantly greater in the NVP-containing arm.
In conclusion, treatment with the NRTI-containing regimen LPV/r+ZDV/3TC, as opposed to the NRTI-sparing regimen LPV/r+NVP, resulted in a 25% decrease in insulin-mediated peripheral glucose disposal and a 22% increase in fasting lipolysis after only 3 months, in the absence of discernable changes in body composition. These findings suggest that certain NRTI may directly contribute to ART-associated disturbances in glucose metabolism, the precise underlying mechanism of which remains to be elucidated but could include NRTI-induced mitochondrial dysfunction. Follow-up of participants in our trial is ongoing and will need to determine to what extent these regimens will continue to differ with respect to their effects on peripheral insulin sensitivity and, over the longer term, potentially also on other aspects of glucose metabolism, body fat distribution and lipid abnormalities. Meanwhile avoiding thymidine analogue NRTI might be appropriate, particularly in patients with (a predisposition to develop) diabetes mellitus.
We are indebted to Katja Tuominen, Rob Simonse, Gerrit-Jan Ilbrink and Ingrid Knufman for their great help during the clamps and their assistance in planning all study days. We would like to thank An Ruiter, Barbara Voermans and Erik Endert from the department of Clinical Chemistry, Laboratory of Endocrinology and Radiochemistry for excellent analytical assistance.
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