PMID- 22590537
OWN - NLM
STAT- MEDLINE
DCOM- 20120919
LR  - 20211021
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 7
IP  - 5
DP  - 2012
TI  - A genome-wide siRNA screen to identify modulators of insulin sensitivity and 
      gluconeogenesis.
PG  - e36384
LID - 10.1371/journal.pone.0036384 [doi]
LID - e36384
AB  - BACKGROUND: Hepatic insulin resistance impairs insulin's ability to suppress 
      hepatic glucose production (HGP) and contributes to the development of type 2 
      diabetes (T2D). Although the interests to discover novel genes that modulate 
      insulin sensitivity and HGP are high, it remains challenging to have a human cell 
      based system to identify novel genes. METHODOLOGY/PRINCIPAL FINDINGS: To identify 
      genes that modulate hepatic insulin signaling and HGP, we generated a human cell 
      line stably expressing beta-lactamase under the control of the human 
      glucose-6-phosphatase (G6PC) promoter (AH-G6PC cells). Both beta-lactamase 
      activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a 
      combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene 
      High-Throughput-Genomics assay was developed to concomitantly measure G6PC and 
      pyruvate-dehydrogenase-kinase-4 (PDK4) mRNA levels. Using this assay, we screened 
      an siRNA library containing pooled siRNA targeting 6650 druggable genes and 
      identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA 
      levels. Pathway analysis indicated that siRNA-mediated knockdown (KD) of genes 
      known to positively or negatively affect insulin signaling increased or decreased 
      G6PC mRNA expression, respectively, thus validating our screening platform. A 
      subset of 270 primary screen hits was selected and 149 hits were confirmed by 
      target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC 
      expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes 
      decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role 
      in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the 
      above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting 
      that they suppress gluconeogenic gene by enhancing insulin signaling. 
      CONCLUSIONS/SIGNIFICANCE: These results support the proposition that the proteins 
      encoded by the genes identified in our cell-based druggable genome siRNA screen 
      hold the potential to serve as novel pharmacological targets for the treatment of 
      T2D.
FAU - Yang, Ruojing
AU  - Yang R
AD  - Department of Metebolic Disorders-Diabetes, Merck Research Laboratories, Rahway, 
      New Jersey, United States of America. ruojing_yang@merck.com
FAU - Lacson, Raul G
AU  - Lacson RG
FAU - Castriota, Gino
AU  - Castriota G
FAU - Zhang, Xiaohua D
AU  - Zhang XD
FAU - Liu, Yaping
AU  - Liu Y
FAU - Zhao, Wenqing
AU  - Zhao W
FAU - Einstein, Monica
AU  - Einstein M
FAU - Camargo, Luiz Miguel
AU  - Camargo LM
FAU - Qureshi, Sajjad
AU  - Qureshi S
FAU - Wong, Kenny K
AU  - Wong KK
FAU - Zhang, Bei B
AU  - Zhang BB
FAU - Ferrer, Marc
AU  - Ferrer M
FAU - Berger, Joel P
AU  - Berger JP
LA  - eng
PT  - Journal Article
DEP - 20120509
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (RNA, Small Interfering)
SB  - IM
MH  - Cell Line, Tumor
MH  - *Diabetes Mellitus, Type 2/genetics/metabolism
MH  - *Genome, Human
MH  - Genome-Wide Association Study
MH  - Genomics/methods
MH  - Gluconeogenesis/*genetics
MH  - Humans
MH  - Insulin Resistance/*genetics
MH  - Liver/*metabolism
MH  - *RNA, Small Interfering
PMC - PMC3348929
COIS- Competing Interests: The authors have the following competing interest: All 
      authors were employees of Merck Research Laboratories at the time of the study. 
      There are no patents, products in development or marketed products to declare. 
      This does not alter the authors' adherence to all the PLoS ONE policies on 
      sharing data and materials, as detailed online in the guide for authors.
EDAT- 2012/05/17 06:00
MHDA- 2012/09/20 06:00
PMCR- 2012/05/09
CRDT- 2012/05/17 06:00
PHST- 2011/11/17 00:00 [received]
PHST- 2012/03/30 00:00 [accepted]
PHST- 2012/05/17 06:00 [entrez]
PHST- 2012/05/17 06:00 [pubmed]
PHST- 2012/09/20 06:00 [medline]
PHST- 2012/05/09 00:00 [pmc-release]
AID - PONE-D-11-23007 [pii]
AID - 10.1371/journal.pone.0036384 [doi]
PST - ppublish
SO  - PLoS One. 2012;7(5):e36384. doi: 10.1371/journal.pone.0036384. Epub 2012 May 9.