Retroviral protease inhibitors used as therapy for HIV-1 infection have been causally associated with serious metabolic side effects, including peripheral lipodystrophy, hyperlipidemia, insulin resistance, and in some cases, overt type 2 diabetes. The etiology of this characteristic clinical syndrome remains unknown. We demonstrate that the HIV protease inhibitor, indinavir, dramatically inhibits insulin-stimulated glucose uptake in 3T3-L1 adipocytes in a dose-dependent manner (63% inhibition observed with 100 M indinavir). Indinavir treatment did not affect early insulin signaling events or the translocation of intracellular Glut1 or Glut4 glucose transporters to the cell surface. To determine whether indinavir may be directly affecting the intrinsic transport activity of glucose transporters, the Glut1 and Glut4 isoforms were heterologously expressed and analyzed in Xenopus laevis oocytes. Indinavir at 100 M had no effect on Glut1 transport activity in Xenopus oocytes, whereas Glut4 activity was significantly inhibited (45% inhibition). Similar effects on glucose transport were observed for other HIV protease inhibitors. We conclude that HIV protease inhibitors as a class are capable of selectively inhibiting the transport function of Glut4 and that this effect may be responsible for a major iatrogenic complication frequently observed in HIV patients.

Trehalose is a naturally occurring disaccharide that has gained attention for its ability to induce cellular autophagy and mitigate diseases related to pathological protein aggregation. Despite decades of ubiquitous use as a nutraceutical, preservative, and humectant, its mechanism of action remains elusive. Here, we showed that trehalose inhibited members of the SLC2A (also known as GLUT) family of glucose transporters. Trehalose-mediated inhibition of glucose transport induced AMPK (adenosine 5′-monophosphate-activated protein kinase)-dependent autophagy regression of hepatic steatosis in vivo, and a reduction in the accumulation of lipid droplets in primary murine hepatocyte cultures. Our data indicated that, by inhibiting glucose transport, trehalose triggers beneficial cellular autophagy.

The liver responds to injury with regulated tissue regeneration. During early regeneration, the liver accumulates fat. Neither the mechanisms responsible for nor the functional significance of this transient steatosis have been determined. In this study, we examined patterns of gene expression associated with hepatic fat accumulation in regenerating liver and tested the hypothesis that disruption of hepatic fat accumulation would be associated with impaired hepatic regeneration. First, microarray-based gene expression analysis revealed that several genes typically induced during adipocyte differentiation were specifically upregulated in the regenerating liver prior to peak hepatocellular fat accumulation. These observations suggest that hepatic fat accumulation is specifically regulated during liver regeneration. Next, 2 methods were employed to disrupt hepatocellular fat accumulation in the regenerating liver. Because exogenous leptin supplementation reverses hepatic steatosis in leptin-deficient mice, the effects of leptin supplementation on liver regeneration in wildtype mice were examined. The data showed that leptin supplementation resulted in suppression of hepatocellular fat accumulation and impairment of hepatocellular proliferation during liver regeneration. Second, because glucocorticoids regulate cellular fat accumulation during adipocyte differentiation, the effects of hepatocyte-specific disruption of the glucocorticoid receptor were similarly evaluated. The results showed that hepatic fat accumulation and hepatocellular proliferation were also suppressed in mice with liver specific disruption of glucocorticoid receptor. In conclusion, suppression of hepatocellular fat accumulation is associated with impaired hepatocellular proliferation following partial hepatectomy, indicating that hepatocellular fat accumulation is specifically regulated during and may be essential for normal liver regeneration. (HEPATOLOGY 2004;40:1322-1332 T he liver regenerates in response to a variety of injuries. [1][2][3] The rodent partial hepatectomy model has been a useful tool with which to investigate the signals that regulate this regenerative response. Following partial hepatectomy, most of the remaining quiescent hepatocytes in the remnant liver tissue quickly proliferate leading to rapid restoration of appropriate liver mass. 4 Analyses of genetically and pharmacologically manipulated mice using this model have begun to identify the coordinated signaling events that regulate hepatic regeneration. These signals include activation of tumor necrosis factor ␣ (TNF␣) interleukin (IL)-6 signaling, 5-7 generation of mitochondrial reactive oxygen species 8 and prostaglandins 9 , and activation of stress-and mitogenactivated-protein kinase cascades. 10 These signals lead to activation of nuclear factor B (NF B) and other transcription factors, which direct an immediate early gene expression program culminating in growth factor-dependent hepatocellular reentry into and progression through the cell cycle [11][12][13] . Once t...

Indinavir appears to be a relatively selective inhibitor of the Glut4 isoform. As the concentration required to significantly inhibit insulin-stimulated glucose uptake in primary rat adipocytes is well within the physiologic range achieved in therapy, we conclude that direct inhibition of Glut4 contributes to the insulin resistance observed in patients receiving this drug.

Trehalose is a disaccharide demonstrated to mitigate disease burden in multiple murine neurodegenerative models. We recently revealed that trehalose rapidly induces hepatic autophagy and abrogates hepatic steatosis by inhibiting hexose transport via the SLC2A family of facilitative transporters. Prior studies, however, postulate that intracellular trehalose is sufficient to induce cellular autophagy. The objective of the current study was to identify the means by which trehalose accesses the hepatocyte cytoplasm, and define the distal signaling mechanisms by which trehalose induces autophagy. We provide gas chromatographic/mass spectrometric, fluorescence microscopic and radiolabeled uptake evidence that trehalose traverses the plasma membrane via SLC2A8 (GLUT8), a homolog of the trehalose transporter-1 (Tret1). Moreover, GLUT8-deficient hepatocytes and GLUT8-deficient mice exposed to trehalose resisted trehalose-induced AMP-activated protein kinase (AMPK) phosphorylation and autophagic induction in vitro and in vivo. Although trehalose profoundly attenuated mTORC1 signaling, trehalose-induced mTORC1 suppression was insufficient to activate autophagy in the absence of AMPK or GLUT8. Strikingly, transient, heterologous Tret1 overexpression reconstituted autophagic flux and AMPK signaling defects in GLUT8-deficient hepatocyte cultures. Together, these data suggest that cytoplasmic trehalose access is carrier-mediated, and that GLUT8 is a mammalian trehalose transporter required for hepatocyte trehalose-induced autophagy and signal transduction.

HIV protease inhibitors (PIs) acutely and reversibly inhibit the insulin-responsive glucose transporter Glut 4, leading to peripheral insulin resistance and impaired glucose tolerance. Minimal modeling analysis of glucose tolerance tests on PI-treated patients has revealed an impaired insulin secretory response, suggesting additional pancreatic ␤-cell dysfunction. To determine whether ␤-cell function is acutely affected by PIs, we assayed glucose-stimulated insulin secretion in rodent islets and the insulinoma cell line MIN6. Insulin release from MIN6 cells and rodent islets was significantly inhibited by the PI indinavir with IC 50 values of 1.1 and 2.1 mol/l, respectively. The uptake of 2-deoxyglucose in MIN6 cells was similarly inhibited (IC 50 of 2.0 mol/ l), whereas glucokinase activity was unaffected at drug levels as high as 1 mmol/l. Glucose utilization was also impaired at comparable drug levels. Insulin secretogogues acting downstream of glucose transport mostly reversed the indinavir-mediated inhibition of insulin release in MIN6 cells. Intravenous infusion of indinavir during hyperglycemic clamps on rats significantly suppressed the first-phase insulin response. These data suggest that therapeutic levels of PIs are sufficient to impair glucose sensing by ␤-cells. Thus, together with peripheral insulin resistance, ␤-cell dysfunction likely contributes to altered glucose homeostasis associated with highly active antiretroviral therapy. Diabetes 52: 1695-1700, 2003

The use of HIV protease inhibitors (PIs) has been associated with several metabolic changes, including lipodystrophy, hyperlipidemia, and insulin resistance. The etiology of these adverse effects remains unknown. PIs have recently been found to cause acute and reversible inhibition of GLUT4 activity in vitro. To determine the acute in vivo effects of indinavir on whole-body glucose homeostasis, glucose tolerance tests were performed on PI-naïve Wistar rats immediately after a single intravenous dose of indinavir. Glucose and insulin levels were significantly elevated in indinavirtreated versus control rats (P < 0.05) during the initial 30 min of the glucose tolerance test. Under euglycemichyperinsulinemic clamp conditions, indinavir treatment acutely reduced the glucose infusion rate required to maintain euglycemia by 18 and 49% at indinavir concentrations of 14 and 27 mol/l, respectively. Muscle 2-deoxyglucose uptake was similarly reduced under these conditions. Restoration of insulin sensitivity was observed within 4 h after stopping the indinavir infusion. Indinavir did not alter the suppression of hepatic glucose output under hyperinsulinemic conditions. These data demonstrate that indinavir causes acute and reversible changes in whole-body glucose homeostasis in rats and support the contribution of GLUT4 inhibition to the development of insulin resistance in patients treated with PIs. Diabetes 51:937-942, 2002

The early growth response-1 transcription factor (Egr-1) is induced as part of the immediate-early gene expression response during early liver regeneration. In the studies reported here the functional significance of EGR-1 expression during liver regeneration was examined by characterizing the hepatic regenerative response to partial hepatectomy in Egr-1 null mice. The results of these studies showed that liver regeneration in Egr-1 null mice is impaired. Although activation of interleukin-6-STAT3 signaling, regulation of expression of hepatic C/ebp␣, C/ebp␤, cyclin D, and cyclin E and progression through the first wave of hepatocellular DNA synthesis occurred appropriately following partial hepatectomy in Egr-1 null mice, subsequent signaling events and cell cycle progression after the first round of DNA synthesis were deranged. This derangement was characterized by increased activation of the p38 mitogen-activated protein kinase and inhibition of hepatocellular metaphase-to-anaphase mitotic progression. Together these observations suggest that EGR-1 is an important regulator of hepatocellular mitotic progression. In support of this, microarray-based gene expression analysis showed that induction of expression of the cell division cycle 20 gene (Cdc20), a key regulator of the mitotic anaphase-promoting complex, is significantly reduced in Egr-1 null mice. Taken together these data define a novel functional role for EGR-1 in regulating hepatocellular mitotic progression through the spindle assembly checkpoint during liver regeneration.