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Muscle-specific knock-out of NUAK family SNF1-like kinase 1 (NUAK1) prevents high fat diet-induced glucose intolerance.

Inazuka F, Sugiyama N, Tomita M, Abe T, Shioi G, Esumi H - J. Biol. Chem. (2012)

Bottom Line: Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype.Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle.Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Japan.

ABSTRACT
NUAK1 is a member of the AMP-activated protein kinase-related kinase family. Recent studies have shown that NUAK1 is involved in cellular senescence and motility in epithelial cells and fibroblasts. However, the physiological roles of NUAK1 are poorly understood because of embryonic lethality in NUAK1 mice. The purpose of this study was to elucidate the roles of NUAK1 in adult tissues. We determined the tissue distribution of NUAK1 and generated muscle-specific NUAK1 knock-out (MNUAK1KO) mice. For phenotypic analysis, whole body glucose homeostasis and muscle glucose metabolism were examined. Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype. Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle. On a high fat diet, MNUAK1KO mice had a lower fasting blood glucose level, greater glucose tolerance, higher insulin sensitivity, and higher concentration of muscle glycogen than control mice. Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle. Consistent with this, insulin signaling was enhanced in the soleus muscle of MNUAK1KO mice, as evidenced by increased phosphorylation of IRS1 Tyr-608, AKT Thr-308, and TBC1D4 Thr-649. These observations suggest that a physiological role of NUAK1 is to suppress glucose uptake through negative regulation of insulin signaling in oxidative muscle.

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NUAK1 is preferentially expressed in highly oxidative tissues.A, quantitative RT-PCR analysis of Nuak1 and Nuak2. RNA was isolated from 10-week-old C57BL/6 mice. The data are the means ± S.E. (n = 3). B, immunoblotting analysis of NUAK1 and LKB1 in heart, Sol, TA, and EDL muscles. Actin was used as a loading control. Protein extracts were from three individual 10-week-old C57BL/6 mice. C, schematic representation of floxed and knock-out alleles of Nuak1. The black arrowheads represent loxP sites. Gray arrows indicate PCR primers P1 and P2 used for the genotyping. Amplification with the P1 and P2 primers yields the 1166-bp product from the floxed allele and the 378-bp product from the knock-out allele. D, genotyping of Nuak1 floxed mice without (control) or with (MNUAK1KO) the Mck-Cre transgene. M, molecular marker. E, real time RT-PCR for Nuak1 mRNA in muscles. RNA was isolated from 10-week-old control and MNUAK1KO mice. The mRNA levels are expressed relative to that in soleus muscle of control mice. The data are the means ± S.E. (n = 3). *, p < 0.05; **, p < 0.01 (Student's t test). F, immunoblot analysis of NUAK1 protein in muscles. Protein extracts of each type of muscle were prepared from three individual mice. Sol, soleus; Quad, quadriceps; Cont, control; MNKO, MNUAK1KO.
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Figure 1: NUAK1 is preferentially expressed in highly oxidative tissues.A, quantitative RT-PCR analysis of Nuak1 and Nuak2. RNA was isolated from 10-week-old C57BL/6 mice. The data are the means ± S.E. (n = 3). B, immunoblotting analysis of NUAK1 and LKB1 in heart, Sol, TA, and EDL muscles. Actin was used as a loading control. Protein extracts were from three individual 10-week-old C57BL/6 mice. C, schematic representation of floxed and knock-out alleles of Nuak1. The black arrowheads represent loxP sites. Gray arrows indicate PCR primers P1 and P2 used for the genotyping. Amplification with the P1 and P2 primers yields the 1166-bp product from the floxed allele and the 378-bp product from the knock-out allele. D, genotyping of Nuak1 floxed mice without (control) or with (MNUAK1KO) the Mck-Cre transgene. M, molecular marker. E, real time RT-PCR for Nuak1 mRNA in muscles. RNA was isolated from 10-week-old control and MNUAK1KO mice. The mRNA levels are expressed relative to that in soleus muscle of control mice. The data are the means ± S.E. (n = 3). *, p < 0.05; **, p < 0.01 (Student's t test). F, immunoblot analysis of NUAK1 protein in muscles. Protein extracts of each type of muscle were prepared from three individual mice. Sol, soleus; Quad, quadriceps; Cont, control; MNKO, MNUAK1KO.

Mentions: To determine the tissue distribution of NUAK1, the level of mouse Nuak1 mRNA in various organs and tissues was measured using quantitative RT-PCR. The distribution of Nuak2 mRNA was also examined because NUAK1 and NUAK2 may have redundant functions (7, 30). As shown in Fig. 1A, Nuak1 was most abundant in the cerebrum and heart, which is consistent with the distribution of human NUAK1 (31). We found that among the skeletal muscles examined, Nuak1 was selectively expressed in the soleus, at a level comparable with the heart. In contrast, Nuak2 was barely detectable in the skeletal muscles and was highly expressed in kidney.


Muscle-specific knock-out of NUAK family SNF1-like kinase 1 (NUAK1) prevents high fat diet-induced glucose intolerance.

Inazuka F, Sugiyama N, Tomita M, Abe T, Shioi G, Esumi H - J. Biol. Chem. (2012)

NUAK1 is preferentially expressed in highly oxidative tissues.A, quantitative RT-PCR analysis of Nuak1 and Nuak2. RNA was isolated from 10-week-old C57BL/6 mice. The data are the means ± S.E. (n = 3). B, immunoblotting analysis of NUAK1 and LKB1 in heart, Sol, TA, and EDL muscles. Actin was used as a loading control. Protein extracts were from three individual 10-week-old C57BL/6 mice. C, schematic representation of floxed and knock-out alleles of Nuak1. The black arrowheads represent loxP sites. Gray arrows indicate PCR primers P1 and P2 used for the genotyping. Amplification with the P1 and P2 primers yields the 1166-bp product from the floxed allele and the 378-bp product from the knock-out allele. D, genotyping of Nuak1 floxed mice without (control) or with (MNUAK1KO) the Mck-Cre transgene. M, molecular marker. E, real time RT-PCR for Nuak1 mRNA in muscles. RNA was isolated from 10-week-old control and MNUAK1KO mice. The mRNA levels are expressed relative to that in soleus muscle of control mice. The data are the means ± S.E. (n = 3). *, p < 0.05; **, p < 0.01 (Student's t test). F, immunoblot analysis of NUAK1 protein in muscles. Protein extracts of each type of muscle were prepared from three individual mice. Sol, soleus; Quad, quadriceps; Cont, control; MNKO, MNUAK1KO.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: NUAK1 is preferentially expressed in highly oxidative tissues.A, quantitative RT-PCR analysis of Nuak1 and Nuak2. RNA was isolated from 10-week-old C57BL/6 mice. The data are the means ± S.E. (n = 3). B, immunoblotting analysis of NUAK1 and LKB1 in heart, Sol, TA, and EDL muscles. Actin was used as a loading control. Protein extracts were from three individual 10-week-old C57BL/6 mice. C, schematic representation of floxed and knock-out alleles of Nuak1. The black arrowheads represent loxP sites. Gray arrows indicate PCR primers P1 and P2 used for the genotyping. Amplification with the P1 and P2 primers yields the 1166-bp product from the floxed allele and the 378-bp product from the knock-out allele. D, genotyping of Nuak1 floxed mice without (control) or with (MNUAK1KO) the Mck-Cre transgene. M, molecular marker. E, real time RT-PCR for Nuak1 mRNA in muscles. RNA was isolated from 10-week-old control and MNUAK1KO mice. The mRNA levels are expressed relative to that in soleus muscle of control mice. The data are the means ± S.E. (n = 3). *, p < 0.05; **, p < 0.01 (Student's t test). F, immunoblot analysis of NUAK1 protein in muscles. Protein extracts of each type of muscle were prepared from three individual mice. Sol, soleus; Quad, quadriceps; Cont, control; MNKO, MNUAK1KO.
Mentions: To determine the tissue distribution of NUAK1, the level of mouse Nuak1 mRNA in various organs and tissues was measured using quantitative RT-PCR. The distribution of Nuak2 mRNA was also examined because NUAK1 and NUAK2 may have redundant functions (7, 30). As shown in Fig. 1A, Nuak1 was most abundant in the cerebrum and heart, which is consistent with the distribution of human NUAK1 (31). We found that among the skeletal muscles examined, Nuak1 was selectively expressed in the soleus, at a level comparable with the heart. In contrast, Nuak2 was barely detectable in the skeletal muscles and was highly expressed in kidney.

Bottom Line: Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype.Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle.Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Japan.

ABSTRACT
NUAK1 is a member of the AMP-activated protein kinase-related kinase family. Recent studies have shown that NUAK1 is involved in cellular senescence and motility in epithelial cells and fibroblasts. However, the physiological roles of NUAK1 are poorly understood because of embryonic lethality in NUAK1 mice. The purpose of this study was to elucidate the roles of NUAK1 in adult tissues. We determined the tissue distribution of NUAK1 and generated muscle-specific NUAK1 knock-out (MNUAK1KO) mice. For phenotypic analysis, whole body glucose homeostasis and muscle glucose metabolism were examined. Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype. Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle. On a high fat diet, MNUAK1KO mice had a lower fasting blood glucose level, greater glucose tolerance, higher insulin sensitivity, and higher concentration of muscle glycogen than control mice. Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle. Consistent with this, insulin signaling was enhanced in the soleus muscle of MNUAK1KO mice, as evidenced by increased phosphorylation of IRS1 Tyr-608, AKT Thr-308, and TBC1D4 Thr-649. These observations suggest that a physiological role of NUAK1 is to suppress glucose uptake through negative regulation of insulin signaling in oxidative muscle.

Show MeSH
Related in: MedlinePlus