Our results show that a single oral dose of the HIV PI indinavir induced insulin resistance in healthy HIV-negative volunteers. The onset of insulin resistance was rapid and occurred at plasma concentrations of indinavir approximating steady-state levels observed in HIV-infected patients maintained on standard clinical doses of this agent [15
]. The magnitude of the decline in insulin-stimulated glucose disposal was approximately 34% and consistent in all subjects. Moreover, most of this reduction in glucose utilization was accounted for by a significant reduction in the rate of non-oxidative glucose disposal suggesting decreased glucose storage. The present finding, therefore, is compatible with the hypothesis that the first event leading to the development of impaired glucose tolerance and type II diabetes in patients treated with the PI indinavir is insulin resistance in skeletal muscle and adipocytes.
Muscle cells and adipocytes are the primary sites for insulin-stimulated glucose disposal [16
]. In response to insulin signaling, specialized glucose transporter vesicles are translocated from intracellular sites to the plasma membrane, thereby facilitating transport of glucose into the cells [17
]. GLUT-4 is the main insulin-responsive glucose transporter in both muscle and adipose tissue. Intracellular transport of glucose by GLUT-4 is a rate-limiting step in insulin-stimulated glucose disposal and the abundance of GLUT-4 in different muscle types correlates roughly with the ability of those muscles to take up glucose [19
studies of the effect of PI on glucose uptake in 3T3-L1 adipocytes and skeletal muscle have shown that various PI including indinavir, amprenavir, ritonavir [8
] and nelfinavir [9
] when added at close to peak therapeutic concentration of 10 μM or greater, cause inhibition of insulin-stimulated glucose uptake. For example, at this concentration, indinavir caused a 26% inhibition of glucose uptake within minutes of addition to the culture medium [8
]. Moreover, indinavir at 5 μM decreased both insulin- and contraction-stimulated glucose transport by average of 40% in isolated rat skeletal muscle [9
The effect on insulin-stimulated glucose transport into muscle is also rapid and detectable after a 4-h incubation in the presence of indinavir (shorter incubation times were not tested). The 40% inhibition at 5 μM in isolated muscle is comparable to the 34% decline in glucose disposal we observed in the present study after a 1200 mg oral dose, and the 20% decrease reported by us previously with a dose of 800 mg three times daily [7
]. Our findings in humans, therefore, are consistent and compatible with the observations made in vitro
The concentrations of indinavir at which we conducted our study (mean Cmax
9.4 μM, AUC 13.5 μM · h) closely resemble those observed in pharmacokinetic studies of healthy volunteers (Cmax
, 11.7 μM; AUC, 23.15 μM · h) [20
]. Drug concentrations are important and may account for some of the discrepancies among published studies. For example, in vitro
studies using indinavir at near-therapeutic concentrations of 10 μM or less for short incubations times (1 h) in both cultured cells [8
] and isolated tissue [9
] suggest that the mechanism of this effect is due directly to rapid blockade of the GLUT-4 transporters. At these concentrations, indinavir caused no defects in signaling pathways, particularly the phosphoinositide-3 kinase or protein kinase B phosphorylation in 3T3-L1 preadipocytes. A similar finding was reported by Caron et al.
using 3T3-F442 preadiocytes, where incubation for up to 8 days in the presence of indinavir at 15 μM (10 μg/ml) did not alter tyrosine phosphorylation [21
]. However, when used at supra-therapeutic concentrations of 100 μM for 48 h, indinavir impaired insulin signaling in HepG2 heptaoma cells [22
]. Similarly another PI, nelfinavir, impaired insulin stimulation of protein kinase B phosphorylation [23
] in vitro
but only after 18 h of incubation at concentrations of ≥ 20 μM, nearly fourfold its Cmax
of 5.2–5.6 μM in humans [24
]. This effect was not observed at lower nelfinavir concentrations (≤ 10 μM) that are in the therapeutic range.
Our findings in human volunteers confirm that the metabolic defect caused by indinavir is rapid and detectable within minutes of achieving standard pharmacologic plasma concentrations of the drug. This strongly suggests a direct mechanism rather than a secondary effect on insulin signaling leading to an impaired ability to inhibit lipolysis. Consistent with this theory, we observed that free fatty acid levels were not acutely increased during euglycemic, hyperinsulinemic clamp and the suppressive effects of hyperinsulinemia on free fatty acid levels did not diminish following indinavir administration. This observation is further reinforced by our earlier published finding that free fatty acid levels were not increased even after 4 weeks of indinavir therapy [7
]. Finally, the bulk of the change in glucose disposal rate was due to a decline in the rate of non-oxidative glucose disposal, the latter reflecting an acute decrease in the rate of glucose storage in muscle and adipocytes. Our findings in humans, therefore, are compatible with the hypothesis that the mechanism by which indinavir decreases insulin-stimulated glucose disposal may be a direct block in the uptake of glucose through the GLUT-4 transporter. Clinically, decreases in this order of magnitude in the rate of muscle glycogen synthesis and insulin-stimulated glucose disposal have been shown to contribute significantly to the development and pathophysiology of type II diabetes mellitus [25
Although the first metabolic defect caused by PI to appear is inhibition of insulin-stimulated glucose disposal, probably through a blockade of the GLUT-4 transporter, other metabolic effects cannot be ruled out. For example, long-term exposure (30 days) to indinavir at concentrations of 15 μM inhibits preadipocyte differentiation presumably by altering the nuclear localization of the sterol regulatory element-binding protein-1 [22
]. However, the effects of glucose deprivation due to long-term blockade of GLUT-4 by indinavir in these cell lines remains unclear. Thus, it is conceivable that with long-term exposure, additional metabolic effects beyond the effect on GLUT-4, such as hypertriglyceridemia, adipocyte de-differentiation and changes in body composition, may follow. Future studies are needed to determine the dose dependence and time-course of the effects of indinavir and other PI drugs in humans.
Recent insights in animal nutrition and fuel storage suggest that GLUT-4 may be more than a passive glucose transporter [26
]. The data implicate a role for these transporters in the communication about nutritional status, fuel processing and storage between the muscle and adipose tissue. For example, selective knock-out of the GLUT-4 gene from fat cells results in a degree of insulin resistance similar to that seen with a muscle-specific knock-out [27
]. Furthermore, the expression of this transporter is tissue specific and regulated by dietary intake [28
]. This might explain how GLUT-4 could play an important role in regulating energy storage in adipose tissue. It is not known whether the longer term complications seen in HIV-infected patients (e.g. body fat redistribution) are manifestations of long-term GLUT-4 blockade or are due to other independent mechanisms. However, changes in body fat distribution have been reported in HIV-infected patients not on therapy with a PI, suggesting that blockade of GLUT-4 cannot be the sole mechanism accounting for these changes [29
We have shown that the effect of indinavir on insulin-stimulated glucose disposal in vivo
is rapid and of the same magnitude as that observed after 4 weeks. Recent data from animal studies suggest that this effect on glucose metabolism is also acutely reversible. Rats infused with indinavir developed insulin resistance, which reversed rapidly within 4 h of discontinuation of indinavir [30
]. The rapid onset of the effects of indinavir and other PI have practical implications for studies of PI-induced insulin resistance. If the morning dose of a PI is omitted before measurement of fasting glucose and insulin, a lower effect may be seen, as PI levels will be in the trough range. For PI that are to be taken with meals (e.g. nelfinavir, ritonavir and lopinavir) holding the drug before study may be common. Consistent dosing is needed to understand the comparative effects of PI and timing of PI ingestion may explain some differences between studies. The delay in peak plasma concentration may also contribute to more profound effects seen in certain metabolic studies such as oral glucose tolerance testing, where the peak plasma levels may coincide with the critical 2-h time point. In the present study, we did not assess the acute effects of a single dose of indinavir on more conventional measures of insulin resistance such as fasting glucose and insulin levels. However, we have previously reported induction of insulin resistance using fasting glucose and insulin after 4 weeks of treatment with indinavir at 800 mg three times daily, with dosing before measurements [7
]. In that study, healthy HIV-negative volunteers had an average of 20% decrease in insulin-stimulated glucose disposal. The magnitude of decrease in insulin sensitivity using the clamp method coincided with an approximate 34% increase in fasting insulin levels and a 47% increase in insulin resistance index by homeostasis model assessment.
It should be noted that we have studied only one PI and our data may not be applicable to all drugs in this class. However, in vitro data using other PI suggest that this may represent a common mechanism. We studied men only; metabolic effects might be different in pre-and postmenopausal women, although there is no reason a priori to suspect that this will prove to be the case given the expression of the GLUT-4 gene.
In summary, our study suggests that the earliest and most direct effect of treatment with indinavir on glucose metabolism is a decrease in insulin-stimulated glucose utilization. The effect is rapid and can be detected at pharmacologic plasma concentrations of indinavir well within the therapeutic range commonly observed in patients. The bulk of the change in glucose disposal rate comes from an acute decrease in the rate of non-oxidative glucose disposal (i.e. storage rate). These findings are compatible with the hypothesis that a mechanism by which indinavir causes insulin resistance is by direct blockade of the glucose transporter GLUT-4. Insights from these studies may provide the basis for designing a new generation of PI drugs that do not perturb glucose metabolism. Future human studies are needed to assess whether these effects are common to other PI and how they might contribute to other observed metabolic and body composition changes reported in patients with HIV.