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Agent and cell-type specificity in the induction of insulin resistance by HIV protease inhibitors
  Ronit Ben-Romano a; Assaf Rudich d; Dora Torok d; Sharon Vanounou a; Klaris Riesenberg b; Francisc Schlaeffer b; Amira Klip d; Nava Bashan a,c
To test agent and cell-type specificity in insulin resistance induced by prolonged exposure to HIV protease inhibitors (HPI), and to assess its relation to the direct, short-term inhibition of insulin-stimulated glucose uptake.
Following prolonged (18 h) and short (5-10 min) exposure to HPI, insulin-stimulated glucose transport, protein kinase B (PKB) phosphorylation, and GLUT4 translocation were evaluated in 3T3-L1 adipocytes, fibroblasts, L6 myotubes, and L6 cells overexpressing a myc tag on the first exofacial loop of GLUT4 or GLUT1.
Prolonged exposure of 3T3-L1 adipocytes to nelfinavir, but not to indinavir or saquinavir, resulted in increased basal lipolysis but decreased insulin-stimulated glucose transport and PKB phosphorylation. In addition, impaired insulin-stimulated glucose uptake and PKB phosphorylation were also observed in the skeletal muscle cell line L6, and in 3T3-L1 fibroblasts. Interestingly, this coincided with increased basal glucose uptake as well as with elevated total-membrane glucose transporter GLUT1 protein content. In contrast to these unique effects of nelfinavir, the mere presence of any of the agents in the 5 min transport assay inhibited insulin-stimulated glucose-uptake activity. This appeared to be caused by direct and specific interaction of the drugs with GLUT4 fully assembled at the plasma membrane, since insulin-stimulated cell-surface exposure of an exofacial myc epitope on GLUT4 was normal.
Independent mechanisms for HPI-induced insulin resistance exist: prolonged exposure to nelfinavir interferes with insulin signaling and alters cellular metabolism of adipocytes and muscle cells, whereas a direct inhibitory effect on insulin-stimulated glucose uptake may occurs through specific interaction of HPI with GLUT4.
Adipose tissue is a key target for HPI in inducing insulin resistance, as the metabolic syndrome described in patients taking HAART consists of gross alterations in fat tissue distribution and dyslipidemia, including elevated circulating FFA and glycerol levels [8]. Adipose tissue is now believed to play a major role in the pathogenesis of systemic insulin resistance. In particular, FFA and various adipose-derived peptides (adipokines) have been identified as modulators of whole-body insulin sensitivity [43-46]. The systemic insulin resistance seen in patients taking HAART also involves impaired insulin responsiveness in skeletal muscle and liver [12]. Consequently, whether these are secondary to the alterations induced by HPI in adipose tissue or represent direct effect(s) of HPI in different tissues is of major interest.
The present study investigated the agent and cell-type specificity of HPI in inducing insulin resistance, focusing on two potential cellular mechanisms. The data presented demonstrate that, while nelfinavir, indinavir and saquinavir can directly inhibit glucose uptake when present acutely following insulin stimulation in cells expressing GLUT4, they manifest differently when used in a prolonged incubation period. Under the prolonged conditions used, only nelfinavir elevated basal lipolysis, increased basal glucose uptake associated with elevated GLUT1 expression, and impaired insulin-stimulated glucose uptake and PKB phosphorylation. These effects of prolonged exposure to nelfinavir could be observed in differentiated 3T3-L1 adipocytes, 3T3-L1 pre-adipocytes and in L6 myotubes. Therefore, HPI at the same concentrations that cause insulin resistance and metabolic alterations in adipocytes, also affects non-adipocyte cells including skeletal muscle cells. It is, therefore, conceivable that the reported HPI-induced insulin resistance of skeletal muscle and liver [12] may represent a combination of direct effects of HPI in these tissues and actions secondary to adipose tissue alterations.
At the cellular level, the mechanism for HPI-induced insulin resistance and lipodystrophy appears to be complex. The current literature suggests the existence of at least three mechanisms: (i) direct inhibition of GLUT4 activity [32, 47]. (ii) activation of basal lipolysis [34, 36, 48] and inhibition of insulin signals towards GLUT4 translocation [34], and (iii) interference with pre-adipocyte to adipocyte differentiation [27-30]. The three mechanisms vary for each agent and time of exposure to the drug. The direct inhibition of GLUT4 activity is common to all HPI tested ([32] and Figure 2, Figure 3) and occurs within minutes, even when the agents are present after insulin stimulation and only in the transport solution. This appears to be a direct but reversible interaction between the HPI and the glucose transporters present on the plasma membrane, since this effect is eliminated upon removal of the agents. Our data are consistent with the notion that the GLUT4 isoform is preferentially vulnerable to this acute inhibition by HPI, since this effect was observed only in GLUT4-expressing cells [3T3-L1 adipocytes, wild-type L6 myotubes (Fig. 2) and L6-GLUT4myc (Fig. 3a)]. In contrast, cells lacking GLUT4 and expressing GLUT1myc were resistant to acute inhibition of glucose-uptake activity by HPI (Fig. 3b). Moreover, by assessing GLUT4 translocation through the extracellular exposure of the myc epitope in non-permeabilized cells, it is possible to conclude that the acute effect of HPI does not interfere with the normal assembly and full insertion of the transporters at the plasma membrane. Rather, it is likely to represent a direct interaction between GLUT4 epitopes and the HPI. These results suggest that, in vivo, if the effective tissue HPI concentrations are sufficient, GLUT4-expressing cells like adipocytes [32], and skeletal muscle cells (Figure 2, Figure 3 and [49]) may be vulnerable to this direct effect of HPI.
When exposure to nelfinavir was extended to 18 h incubation, 3T3-L1 adipocytes exhibited increased basal lipolysis and elevated basal but reduced insulin-stimulated glucose uptake [34]. These effects, associated with enhanced total membrane GLUT1 expression (Fig. 4) and impaired capacity of insulin to stimulate PKB phosphorylation (Fig. 5), are likely independent of the direct inhibitory effect that HPI drugs exhibit on glucose uptake, for several reasons. First, the fall in insulin-stimulated glucose uptake following prolonged nelfinavir treatment is observed in the absence of the agent in the transport solution. Second, this lessened insulin action is not restricted to GLUT4-expressing cells, since 3T3-L1 pre-adipocytes and L6-GLUT1myc myoblasts also exhibit comparable effects (Figure 4, Figure 5). Third, while prolonged nelfinavir impaired insulin-stimulated PKB phosphorylation (Figure 1, Figure 5) and GLUT4 translocation [34], the short-term effect of HPI did not interfere with PKB phosphorylation [32] nor with GLUT4 translocation ([32] and Fig. 3c).
Since at least the direct inhibition of GLUT4 and the long-term effect of nelfinavir may represent independent cellular mechanisms for insulin resistance, different candidate molecular mediators may be speculated to be involved. While the molecular basis for the direct interaction between HPI and GLUT4 has yet to be identified, several hypotheses may be raised to explain the alterations induced by prolonged exposure to nelfinavir. Increased expression of proteins like SH2-domain-containing 5-phosphatase and/or protein phosphatase 2A, potentially through interference with their normal degradation, could result in altered insulin signaling compatible with the effect of prolonged nelfinavir: impaired PKB activation in the face of normal phosphatidylinositol 3-kinase activation ([34] and unpublished data). Increased cellular cyclic AMP levels would explain elevated lipolysis [50], impaired PKB activation by insulin [51] and increased GLUT1 expression [52, 53]. Finally, HPI-induced inhibition of adipocyte differentiation may involve yet other proteins, such as sterol regulatory element binding protein 1 [29]. It is tempting to speculate that HPI drugs inhibit a mammalian protein degradation system resulting in increased expression of proteins, which, in turn, affects lipolysis, insulin action and differentiation. However, so far, such a mechanism has only been shown for the degradation of apolipoprotein B, potentially explaining HPI-induced dyslipidemia [54].
In conclusion, HPI drugs have the potential to cause insulin resistance and metabolic alterations in various cell types representing targets of insulin action, through diverse and possibly independent mechanisms. Understanding these effects at the cellular and molecular level will be essential for both the design of new HPI with a milder side-effect profile, and the planning of therapeutic strategies to combat the unwanted effects of HAART.
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