The level of monocyte CD36 was also assessed in healthy controls (n = 10) receiving no ARV therapy followed over 4–12 weeks. No significant changes in CD36 levels were observed in controls followed over time (Fig. 1c and Fig. 2c).
Two HIV-infected individuals had elevated fasting triglyceride and cholesterol levels at the time of enrolment and before initiating antiretroviral therapy. Of the remaining eight patients, two developed elevated cholesterol (total cholesterol > 5.5 mmol/l) and/or triglyceride levels (> 2.2 mmol/l) by week 8 of therapy. Both of these individuals had a marked decrease in CD36 levels (> 75% decrease) by day 7 of therapy.
THP-1 and C32 cells treated with 10 μmol/l ritonavir had significantly lower CD36 levels than appropriate controls, as determined by flow cytometry. The mean log fluorescence over background in THP-1 cells (n = 3) was 4.10 ± 0.87 in ritonavir-treated cells and 5.67 ± 1.09 in untreated cells; the average percentage decrease was 28 ± 2.58% (P < 0.05, paired Student's t-test). The mean log fluorescence over background in C32 cells (n = 5) was 15.68 ± 9.70 in ritonavir-treated cells and 25.35 ± 6.12 in untreated cells; the average percentage decrease was 40.26 ± 14.5% (P < 0.05, paired Student's t-test). Neither nevirapine (data not shown) nor zidovudine treatment of THP-1 cells had a significant effect on CD36 surface levels (mean log fluorescence over background 6.29 ± 0.58 for zidovudine-treated cells and 5.44 ± 1.01 for untreated cells (P = NS).
CD36 plays an important role in FA, lipid and glucose metabolism, and CD36 deficiency has been directly linked to defective fatty acid utilization, dyslipidaemia, and insulin resistance [19–26]. Our results demonstrate that ARV therapy that includes a PI is associated with an early and marked decrease in CD36 levels in healthy and HIV-infected individuals. These in vivo findings are supported by in vitro data showing that ritonavir, but not zidovudine, induces a decrease in CD36 levels in different human cell lines. If CD36 deficiency is sustained in vivo, these observations provide a putative mechanism by which ARV therapy may contribute to insulin resistance and other metabolic complications of lipodystrophy.
CD36 mediates the uptake of modified lipoproteins and has been implicated in both pro- and antiatherogenic processes [28–33]. CD36 is also a major fatty acid transporter in tissues with high metabolic capacity. Using CD36 knockout mice, CD36 was shown to be essential for normal rates of FA uptake and oxidation, mediating 50–80% of uptake in the myocardium, adipose tissue and skeletal muscle in vivo . The phenotype of spontaneous hypertensive rats, which includes dyslipidaemia and features of human insulin resistance syndromes, has been linked to a spontaneous deletion in CD36 . Transgenic rescue of defective CD36 in spontaneous hypertensive rats improved insulin responsiveness, reduced glucose intolerance and lowered levels of serum FA , providing direct evidence that CD36 deficiency contributes to insulin resistance and disordered FA metabolism. Similarly, CD36 null mice have increased serum levels of FA, triglyceride and cholesterol and abnormal glucose and insulin levels .
Based on the rodent models, CD36 deficiency in humans might be expected to result in dyslipidaemia, disrupted PPARγ activation , impaired fat cell differentiation and storage, and decreased insulin responsiveness [19,20]. Although studies are limited, human CD36 deficiency has been associated with elevated serum total cholesterol, low density lipoprotein cholesterol, decreased uptake of modified low density lipoprotein by monocyte-derived macrophages and, most recently, with impaired glucose tolerance, insulin resistance and hyperlipidaemia [25,26,35,36]. CD36 deficiency in humans has also been linked to cardiomyopathies associated with decreased myocardial FA utilization [37,38].
Features of lipodystrophy have been reported in individuals receiving nucleoside reverse transcriptase inhibitors alone, in particular stavudine [9,39]. Brinkman and colleagues have postulated that these changes may be secondary to drug-induced mitochondrial toxicity . In our study, individuals on combination ARV therapy that included a PI and stavudine had more marked decreases in CD36 levels than healthy individuals on PI alone. This may be attributable to HIV infection and/or combination ARV therapy, which may have additive or synergistic effects on CD36 expression and PPAR signalling. HIV infection and ARV therapy may ultimately induce lipodystrophy by a combination of effects on CD36 expression, mitochondrial function and other genes or gene products important in lipid and carbohydrate metabolism. Of note, CD36 deficiency might also contribute to mitochondrial dysfunction since CD36 mediates the uptake of FA essential to cell functions such as mitochondrial β-oxidation and activation of PPARγ [32,39–41].
The molecular mechanism by which ARV therapy induces CD36 deficiency is unknown. Whether decreased CD36 surface levels are the result of altered signalling, reduced activation of the PPARγ–RXR heterodimer  or another mechanism remains to be elucidated. It will be important to determine if PPARγ agonists, which upregulate CD36 , can prevent or reverse the suppression of CD36 and the metabolic alterations induced by ARV drugs and/or PI. Preliminary studies in our laboratory indicate that PPARγ agonists can rescue CD36 deficiency induced by ritonavir in vitro. Furthermore, troglitazone, which upregulates CD36, has recently been reported to improve insulin sensitivity, decrease visceral fat and triglycerides, and increase subcutaneous adipose tissue in a small cohort of HIV-infected patients with ARV therapy-associated lipodystrophy .
Of interest, not all individuals taking ARV drugs or PI in this study showed reduced levels of CD36. Similarly, many but not all HIV-positive patients receiving combination ARV therapy develop lipodystrophy and approximately 70% develop dyslipidaemia at levels associated with increased cardiovascular disease [6,8,9]. It remains to be determined whether the degree of CD36 deficiency we observed is sufficient to mediate the metabolic abnormalities observed in HIV-positive patients receiving combination ARV therapy and if CD36 levels are predictive of these treatment complications. A longitudinal analysis of CD36 expression in monocytes, adipocytes and peripheral muscle in controlled clinical trials will be required to answer these questions definitively.
In summary, ARV therapy including a PI in this study resulted in a marked and early decrease in CD36 in a proportion of recipients. If ARV therapy-induced CD36 deficiency is sustained in vivo, it may contribute to the induction of lipodystrophy and its associated metabolic complications including insulin resistance and dyslipidaemia.
We thank Drs Y. Khaliq and A. MacCarthy at the Ottawa General Hospital for allowing us to study a group of their patients. We thank Dr S. Read and Cheryl Smith for assistance with flow cytometry. We thank Drs J. Brunton and K. MacDonald for helpful suggestions and support and Mrs K. Raposa, study nurse, who was responsible for enrolling patients and collecting blood samples.
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