Mechanism of atorvastatin induced glucose uptake

Group Leader

Univ.-Prof. Dr. März Winfried
(winfried.maerz@klinikum-graz.at)

Department/Institute

Univ. Klinik/KIMCL

Group Members

	Dr. Gruber Hans Jürgen
	Haßlitzer Karin
	Landl Evamaria
	Dr. Mayer Claudia
	Pailer Sabine
	Paulitsch Sabine
	Dr. Scharnagl Hubert
	Dr. Stojakovic Tatjana

Project Duration

01.04.2005 - 01.04.2007

Summary

MECHANISM OF ATORVASTATIN INDUCED GLUCOSE UPTAKE 1. Rationale 1a.) Atorvastatin and glucose metabolism As outlined in our previously proposal ´Effect of atorvastatin on insulin signalling and glucose metabolism´ we hypothesized that statins may, beyond their lipid lowering effects affect positively the metabolism of glucose. Therefore, we investigated the effects of atorvastatin on glucose metabolism by contriving an in vitro glucose kinetic assay. L6 skeletal muscle cells (ATTC CRL-1458) were differentiated to myotubes and treated with various concentrations of atorvastatin [0.3 µM-1 µM] and/or insulin [100 nM]. Glucose uptake was assessed by incubating the treated cells with the metabolically inactive glucose analogue 2-deoxy-D-[1-3]glucose (2-DG). Our results clearly show a beneficial effect of atorvastatin on glucose uptake. At 1µM, atorvastatin produced an increase by 42 % in 2-DG uptake compared to mock cells. The positive control, insulin [100nM] produced an increased 2-DG uptake by 51%. Co incubation of atorvastatin [1µM] and insulin [100nM] resulted in a slight synergistic effect on 2-DG uptake (+61%) compared to mock cells. Cells were additionally incubated with mevalonate [0,5mM] to verify that the effect of atorvastatin on 2-DG uptake was mediated by specific inhibition of HMG CoA reductase. Co- incubation of atorvastatin [1µM] and mevalonate [0,5mM] blunted the increment in 2-DG uptake from 42% to 17%. To further investigate the effect of atorvastatin on glucose uptake, we focused on the main glucose transporter in skeletal muscle cells, GLUT-4. Upon an insulin stimulus GLUT-4 vesicles translocate from the cytosol to the membrane and facilitate the uptake of glucose into the cell. To examine the effect of atorvastatin on GLUT-4 translocation we established an antibody based indirect fluorescent assay. L6 myotubes treated with atorvastatin or insulin were fixed for detection of GLUT-4 with a primary antibody, specific for GLUT-4 and a secondary fluorescent conjugated antibody for detection in a fluorometer. Insulin treatment showed an increase of 24% in GLUT-4 activation compared to untreated cells. Atorvastatin at 1µM increased GLUT-4 translocation up to 19%, and was this was again attenuated by co-incubation with mevalonate. These results suggest that enhanced glucose uptake induced by atorvastatin is mediated, at least in part, by GLUT-4 vesicles. 1b.) Statins and insulin signalling Investigations on possible interactions between statins and the insulin signalling pathway are very limited as only some putative effects are described. Of most interest is the question, whether there is a linkage between the insulin signalling pathway and the atorvastatin induced glucose uptake observed in our experiments, or whether the atorvastatin induced glucose uptake is independently of the insulin signalling pathway. Especially the effects of atorvastatin on glucose metabolism with regard to insulin resistance may become clinically relevant. Furthermore, because we have shown that the glucose transporter GLUT-4 is involved in atorvastatin induced glucose uptake, the phosphoinositide 3 kinase (PI3K) / protein kinase B (PKB) pathway, which activates GLUT-4 may be a possible target for atorvastatin. Dimmeler et al. showed that statins are able to activate the PI3K/PKB pathway. Especially the PKB is known to transduce to and cross talk with other signalling pathways possibly involved in this mechanism, like the MAP Kinase pathway and the Rho GTPase family. 1c.) Statins, insulin signalling and fatty acid metabolism Finally, there is also evidence that statin-mediated effects on the fatty acid metabolism influence the glucose metabolism. The peroxisome proliferator activator protein receptors (PPARs) are known to be major regulators of intra- and extracellular fatty acid metabolism, especially PPAR alpha. PPARs belong to the superfamily of nuclear receptors that are ligand-activated transcription factors. There is preliminary evidence that PPAR alpha activation would also result in improved insulin sensitivity. A possible mechanism for this PPAR alpha activation is the statin induced inhibition of Rho, which results in PPAR alpha activation. To our knowledge, our work is the first which shows a clear beneficial effect of atorvastatin on glucose metabolism. This raises the possibility that atorvastatin might positively affect insulin sensitivity and might indeed help to prevent the transition from impaired glucose tolerance to manifest type 2 diabetes. Before clinical studies of this issue are conducted, we suggest to examine in detail the mechanism of atorvastatin induced glucose uptake in vitro. 2. Objectives The aim of the present study is to investigate the mechanism of the HMG-CoA reductase inhibitor atorvastatin induced glucose uptake and the underlying signalling pathways, especially the insulin signalling pathway. This is achieved by monitoring glucose metabolism kinetics. Further, the effects of the drug on signal transduction pathways, transcription factor activation and resulting gene expression will be investigated.