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. 2013;8(4):e58797.
doi: 10.1371/journal.pone.0058797. Epub 2013 Apr 1.

Insulin-receptor substrate-2 (irs-2) is required for maintaining glucokinase and glucokinase regulatory protein expression in mouse liver

Isabel Roncero  1 , Elvira Alvarez, Carlos Acosta, Carmen Sanz, Pedro Barrio, Veronica Hurtado-Carneiro, Deborah Burks, Enrique Blázquez
Affiliations

Insulin-receptor substrate-2 (irs-2) is required for maintaining glucokinase and glucokinase regulatory protein expression in mouse liver

Isabel Roncero et al. PLoS One. 2013.

Abstract

Insulin receptor substrate (IRS) proteins play important roles in hepatic nutrient homeostasis. Since glucokinase (GK) and glucokinase regulatory protein (GKRP) function as key glucose sensors, we have investigated the expression of GK and GKRP in liver of Irs-2 deficient mice and Irs2(-/-) mice where Irs2 was reintroduced specifically into pancreatic β-cells [RIP-Irs-2/IRS-2(-/-)]. We observed that liver GK activity was significantly lower (p<0.0001) in IRS-2(-/-) mice. However, in RIP-Irs-2/IRS-2(-/-) mice, GK activity was similar to the values observed in wild-type animals. GK activity in hypothalamus was not altered in IRS-2(-/-) mice. GK and GKRP mRNA levels in liver of IRS-2(-/-) were significantly lower, whereas in RIP-Irs-2/IRS-2(-/-) mice, both GK and GKRP mRNAs levels were comparable to wild-type animals. At the protein level, the liver content of GK was reduced in IRS-2(-/-) mice as compared with controls, although GKRP levels were similar between these experimental models. Both GK and GKRP levels were lower in RIP-Irs-2/IRS-2(-/-) mice. These results suggest that IRS-2 signalling is important for maintaining the activity of liver GK. Moreover, the differences between liver and brain GK may be explained by the fact that expression of hepatic, but not brain, GK is controlled by insulin. GK activity was restored by the β-cell compensation in the RIP-Irs-2/IRS-2 mice. Interestingly, GK and GKRP protein expression remained low in RIP-Irs-2/IRS-2(-/-) mice, perhaps reflecting different mRNA half-lives or alterations in the process of translation and post-translational regulation.

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Conflict of interest statement

Competing Interests: This work was supported by grants from MICINN (SAF2006-0475 and SAF2009-11297), Ayudas del Programa de Creación y Consolidación de Grupos de Investigación UCM-Banco Santander (GR58/08, GR35/10A, GR35/10B and GR42/10), Fundación de Investigación Médica Mutua Madrileña and IODURE project, CIBER de Diabetes y Enfermedades Metabólicas Asociadas, an initiative of ISCIII (Ministerio de Ciencia e Innovación). The authors adhere to all the PLOS ONE policies regarding the sharing of data and materials.

Figures

Figure 1
Figure 1. Glucokinase activity in soluble fractions of liver and hypothalamus.
Extracts were prepared from liver and hypothalamus of female mice of the indicated genotypes. GK activity was calculated by subtracting the glucose-phosphorylating activity at 0.3 mM glucose from the total activity measured at 30 mM glucose. The bars represent means ± SE. n = 8 IRS-2(−/−) (6–7 weeks old), n = 10 WT (6–7 weeks old), n = 5 RIP-Irs-2/IRS-2(−/−) at ∼16 months of age, and n = 5 WT (at ∼16 months of age). GK activity in liver from wild type mice (6–7 weeks old) was considered as 1. *** p<0.001 (IRS-2(−/−) vs WT mice).
Figure 2
Figure 2. GK and GKRP mRNA levels in livers from IRS-2(−/−), RIP-Irs-2/IRS-2(−/−) and their wild type mice.
Levels of mRNA encoding GK (A) and GKRP (B) were measured in liver from IRS-2(−/−) or from RIP-Irs-2/IRS-2(−/−) and their wild-type mice. The bars represent means ± SE (n = 8 animals per experimental group). The values obtained in wild type mice were considered as 1. *** p<0.001 (IRS-2(−/−) vs WT mice).
Figure 3
Figure 3. Analysis of GK and GKRP protein expression in liver from IRS-2(−/−), RIP-Irs-2/IRS-2(−/−) and their wild type mice.
Levels of total GK (A) and GKRP (B) were assessed by Western blots of liver lysates prepared from IRS-2(−/−) or from RIP-Irs-2/IRS-2(−/−) and their wild type mice. The bars represent means ± SE of the densitometric values normalized by β-actin (n = 5–8 animals per group). The values obtained in wild type mice were considered as 1. *** p<0.001 (IRS-2(−/−) or RIP-Irs-2/IRS-2(−/−) vs their WT mice). Representative Western blots images are shown.
Figure 4
Figure 4. Analysis of hypothalamic GK and GKRP protein expression in IRS-2(−/−) and wild type mice.
Total protein levels of GK and GKRP in hypothalamus from IRS-2(−/−) and their wild type mice. The bars represent means ± SE of the densitometric values normalized by β-actin (n = 3 animals per group). The values obtained in wild type mice were considered as 1. Representative Western blots images are shown.
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This work was supported by grants from MICINN (SAF2006-0475 and SAF2009-11297), Ayudas del Programa de Creación y Consolidación de Grupos de Investigación UCM-Banco Santander (GR58/08, GR35/10A, GR35/10B and GR42/10), Fundación de Investigación Médica Mutua Madrileña and IODURE project, CIBER de Diabetes y Enfermedades Metabólicas Asociadas, an initiative of ISCIII (Ministerio de Ciencia e Innovación). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.