Dharmacon SMARTpool^® siRNA Reagents identify regulators of insulin-responsive glucose transport- a critical component in the etiology of type 2 diabetes mellitus.

Ciaran J. Faherty, Ph.D.

Diabetes, a metabolic disorder, is a global health concern that is projected to afflict 366 million people by the year 2030[1].  While diabetes is treatable with exogenous insulin hormone that regulates glucose uptake, critical target tissues may become resistant to such treatment [2,3]. Thus, understanding the cause of insulin resistance is of great interest for developing more viable and longer-lasting therapeutic strategies.

Type 2 diabetes mellitus represents ~90-95% of all diabetes cases[4], and is caused by resistance of cells to take up and metabolize insulin and a reduced production of insulin by the pancreas.  Insulin resistance means that cells are unable to transport glucose across the membrane, leading to long term increases in blood glucose level.  This buildup of extracellular glucose can lead to a range of complications such as cardiovascular disease, chronic renal failure and retinal and nerve damage.  Because glucose transport is the rate-limiting step for glucose utilization by target tissues, it is important to fully understand all the factors involved in regulating transport. In particular, one critical component is the insulin-induced GLUT4-mediated translocation of glucose.  GLUT4 is expressed in adipose, cardiac and skeletal muscle cells and is  a key modulator in whole body glucose homeostasis [5].  Loss of the GLUT4 glucose transporter in these cells contributes significantly to insulin resistance and the progression of diabetes.  Therefore identifying factors instrumental in the regulation of glucose transport in adipose tissue will provide therapeutic avenues for treatments involving refractory target tissues with respect to glucose uptake in diabetic patients[6,7].

Recently, major advances that further our understanding of key regulators of insulin-mediated glucose transport in adipose tissue were published in the Proceeding of the National Academy of Sciences[7].  Researchers from the laboratory of Michael P. Czech, at the University of Massachusetts Medical School, utilized RNA interference (RNAi) to decipher the role that protein kinases play in these insulin-dependent processes[7].  To this end, SMARTpool® siRNA reagents from Dharmacon were used to screen for protein kinases expressed in adipocytes that could potentially regulate insulin-responsive glucose transport.    

Insulin enhances GLUT4 recruitment to the plasma membrane for glucose transport, a process that involves other key players including peroxysome proliferator activated receptor (PPARg). The group set about to uncover additional regulators of insulin-mediated glucose uptake.[8]  They concentrated on a set of 58 protein kinases expressed in adipose cells, and then performed an RNAi screen using SMARTpool® siRNA reagents.  The siRNA-mediated knockdown of six of these targets significantly affected the insulin-mediated uptake of tritiated 2-deoxyglucose, the gold-standard for assessing glucose uptake.  Of the six genes identified in the primary screen, five were shown to be negative regulators of insulin-mediated glucose uptake: PCTAIRE-1, PFTAIRE-1, MAP4k4, IKKa, IKKb.  The sixth protein kinase, ILK, appeared to be a positive regulator based on the measurement of glucose uptake following the siRNA-mediated knockdown.  Most importantly, in a follow-up analysis, all six identified hits were confirmed to affect insulin-mediated glucose uptake.

Additional experiments showed that these novel targets directly affected known regulators of insulin-mediated glucose uptake: AKT2, GLUT4, PPARg and/or C/EBP-a. Interestingly, Czech’s group demonstrated that the ability of MAP4k4, a member of the Sterile 20 (Ste20) family of protein kinases, exerts its effect on PPARg and C/EBP-a,  a unique function when compared to 22 other Ste20 kinase family members.  This ability of MAP4k4 may also explain the activity of tumor necrosis factor-alpha (TNF-a) in insulin resistance[9]. 

By applying RNAi technology using SMARTpool^® siRNA reagents, Czech’s group selectively and reproducibly knocked down key protein kinases in adipose cells and identified regulators of insulin-mediated glucose uptake.  In doing so they not only uncovered potential key regulators of this process, but verified the role of previously reported modulators (e.g. IKKb) of the insulin signaling pathway [10].  These results will be instructive in understanding the biochemical response of patients to a variety of potential treatments[11].  More importantly, the identification of both positive and negative regulators of insulin-mediated glucose uptake should lead to the development of new approaches to combat insulin resistance in diabetic patients. 

 

References

1. Wild, S., et al. "Global Prevalence of Diabetes: Estimates for the year 2000 and projections for 2030 " Diabetes Care 27 (2004): 1047-53.

2. Banting, F. G., et al. "The Effect of Pancreatic Extract (Insulin) on Normal Rabbits." Am J Physiol 62 (1922): 162-76.

3. Yu, Y., and H. N. Ginsberg "Adipocyte Signaling and Lipid Homeostasis: Sequelae of Insulin-Resistant Adipose Tissue " Circulation Research 96 (2005): 1042-52.

4. Center for Disease Control and Prevention. Available: http://www.cdc.gov/

5. Olefsky, J. M. "Insulin-stimulated Glucose Transport Minireview Series." J Biol Chem 274.4 (1999): 1863.

6. Abel, E. D., et al. "Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver." Nature 409.6821 (2001): 729-33.

7. Tang, X., et al. "An RNA interference-based screen identifies MAP4K4/NIK as a negative regulator of PPARgamma, adipogenesis, and insulin-responsive hexose transport." Proc Natl Acad Sci U S A 103.7 (2006): 2087-92.

8. Oatley, P. B., et al. "GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein." Biochem J. 327 (1997): 637-42.

9. Gwozdziewiczova, S., et al. "TNF-alpha in the development of insulin resistance and other disorders in metabolic syndrome." Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 149.1 (2005): 109-17.

10. Yuan, M., et al. "Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta." Science 293.5535 (2001): 1673-77.

11. Echeverri, C. J., and N. Perrimon. "High-throughput RNAi screening in cultured cells: a user's guide." Nat Rev Genet 7.5 (2006): 373-84.

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