Authors
Wei Zhang^1,2, Barry J. Thompson³, Ville Hietakangas^4#, Stephen M. Cohen^1,2#

1 - Institute of Molecular and Cell Biology, Singapore, Singapore
2 - Department of Biological Sciences, National University of Singapore, Singapore
3 - London Research Institute, Cancer Research UK, London, United Kingdom
4 - Institute of Biotechnology, University of Helsinki, Helsinki, Finland
# These authors contributed equally to this work

Published in PLoS Genetics December 29, 2011

Abstract
The insulin/IGF-activated AKT signaling pathway plays a crucial role in regulating tissue growth and metabolism in multicellular animals. Although core components of the pathway are well defined, less is known about mechanisms that adjust the sensitivity of the pathway to extracellular stimuli. In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, which is a hallmark of diabetes. Here we present the results of a genetic screen in Drosophila designed to identify regulators of insulin sensitivity in vivo. Components of the MAPK/ERK pathway were identified as modifiers of cellular insulin responsiveness. Insulin resistance was due to downregulation of insulin-like receptor gene expression following persistent MAPK/ERK inhibition. The MAPK/ERK pathway acts via the ETS-1 transcription factor Pointed. This mechanism permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels.

Author Summary: Insulin signaling is an important and conserved physiological regulator of growth, metabolism, and longevity in multicellular animals. Disturbance in insulin signaling is common in human metabolic disorders. For example insulin resistance is a hallmark of diabetes and metabolic syndrome. While the core components of the insulin signaling pathway have been well established, the mechanisms that adjust the insulin responsiveness are only known to a limited extent. Here we present results from a genetic screen in Drosophila that was designed to identify regulators of cellular insulin sensitivity in an in vivo context. Surprisingly, we discovered cross-talk between the epidermal growth factor receptor (EGFR)–activated MAPK/ERK and insulin signaling pathways. This regulatory mechanism, which involves transcriptional control of insulin-like receptor gene, is utilized in vivo to maintain circulating glucose at appropriate levels. We provide evidence for a regulatory feed-forward mechanism that allows for dynamic transient responsiveness as well as more stable, long-lasting modulation of insulin responsiveness by growth factor receptor signaling. The combination of these mechanisms may contribute to robustness, allowing metabolism to be appropriately responsive to physiological inputs while mitigating the effects of biological noise.

Figure Legend : EGFR-MAPK/ERK signaling regulates InR expression and FOXO localization in vivo. (A) Histogram showing the levels of rp49, inr and ksr mRNAs measured by quantitative RT-PCR. Wandering 3rd instar larvae expressed UAS-ksrRNAi under ubiquitous tubulin-Gal4 control. Controls expressed tubulin-Gal4 without the UAS-RNAi transgene. Total RNA was isolated from imaginal discs, fat body, salivary gland and body wall. RNA levels were normalized for cDNA synthesis before Q-PCR. RNA levels were normalized to kinesin mRNA. The efficiency of KSR depletion is shown in dark gray bars. Student’s t-test: (*) p < 0.05. (B) Immunoblot to detect the level of InR protein in fat body from wandering 3rd instar larvae expressed UAS-KSRRNAi under Tubulin-Gal4 control. Samples were run on the same gel, but intervening lanes have been removed as indicated. (C) Left panels: Immunofluorescent images of fat body dissected from wandering 3rd instar larvae stained with anti-FOXO (green). Nuclei were labeled with DAPI (Blue). Larvae expressed UAS-ksrRNAi under pumpless-Gal4 control. Control larvae expressed Gal4 without the RNAi transgene. Right panel: Quantification of the ratio between nuclear FOXO and cytoplasmic FOXO in fat body expressing ppl-Gal4 or with UAS-ksrRNAi. Subcellular regions were defined by DAPI staining. FOXO intensities were measured in pixels from digital images using ImageJ. Error bars represent standard deviation from measurement of at least 24 cells for each genotype. Student’s t-test: (***) p < 0.001. (D) Histogram showing the levels of rp49 and inr mRNAs measured by quantitative RT-PCR. Wandering 3rd instar larvae expressed UAS-dnEGFR under pumpless-Gal4 control. Controls expressed Gal4 without the UAS transgene. Total RNA was isolated from the fat body. RNA levels were normalized to kinesin mRNA. Error bars represent standard deviation from 3 independent experiments. Student’s t-test: (*) p < 0.05. (E) Immunoblot to visualize the level of InR protein in fat body from wandering 3rd instar larvae expressing UAS-dnEGFR under pumpless-Gal4 control. Samples were run on the same gel, but intervening lanes have been removed as indicated.

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